In vitro cell culture technique protocol manual

8/18/2019 In vitro cell culture technique protocol manual

1/48

CHAPTER 1INTRODUCTION TO IN VITRO TOXICOLOGY AND CELL CULTURE In vitro toxicity testing is the scientific analysis of the effects of toxic chemical

substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing

methods are employed primarily to identify potentially hazardous chemicals and/or to

confirm the lack of certain toxic properties in the early stages of the development of

potentially useful new substances such as therapeutic drugs , agricultural chemicals, direct

food additives, cosmetics, etc. In vitro models offer a number of ethical and economic

advantages over animal testing. hese assays are being used earlier in toxicity testing

pipelines, often to determine risk assessment and/or set up controls that will ultimately spare

animal life. he combination of lower!cost and higher throughput assays can help bring

products to market faster. "dditionally, in vitro models give researchers an advantage inunderstanding the biological processes involved in a toxic response sooner than if they were

depending on the visual inspection of a live animal. In vitro techni#ues represent tools that

the toxicologist can utilize at various stages of drug development to design and select

compounds to perform specialized evaluations and to address preclinical or clinical issues

that may arise. he use of in vitro technologies contributes to the scientific #uality and

economy of the safety assessment process as well as to the toxicologists' commitment to the

three $%s of reduction, refinement, and replacement, and a fourth $, responsibility."dvancements in cell culture have given researchers a viable alternative and/or

complement to live animal testing. In vitro investigations in specialized cell cultures have

been used to clarify the mechanism of toxic action of chemicals on a particular target organ.

&ell culture is the techni#ue by which either prokaryotic or eukaryotic cells are grown under

controlled conditions. n practice the term cell culture refers to culturing of cells derived

from multicellular eukaryotes, especially animal cells. he cellular systems used in toxicity

studies include primary cells, genetically modified cells, immortalized cells, stem cells, and

cells in different stages of differentiation and transformation, co!cultures of different cell

types, etc. he focus is on the cellular models used to study chemical toxicity, specific

toxicity end points at levels including molecular, and the extrapolation of data obtained from

in vitro models to the context of in vivo . hus, in vitro toxicology can potentially replace

animal use in toxicological evaluations to a very great extent.

ased on the characteristics of the source of cells, cell cultures are classified as follows*

Primary cell culture * +ne that is started from tissues/organ freshly removed from theorganism. hey are generally prepared by treating the original tissue with cell dispersing

1

http://en.wikipedia.org/wiki/Toxicology_testinghttp://en.wikipedia.org/wiki/Scientifichttp://en.wikipedia.org/wiki/Scientific_methodhttp://en.wikipedia.org/wiki/Toxichttp://en.wikipedia.org/wiki/Chemical_substancehttp://en.wikipedia.org/wiki/Chemical_substancehttp://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/wiki/Mammalianhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Medicationhttp://en.wikipedia.org/wiki/Agriculturalhttp://en.wikipedia.org/wiki/Toxicology_testinghttp://en.wikipedia.org/wiki/Scientifichttp://en.wikipedia.org/wiki/Scientific_methodhttp://en.wikipedia.org/wiki/Toxichttp://en.wikipedia.org/wiki/Chemical_substancehttp://en.wikipedia.org/wiki/Chemical_substancehttp://en.wikipedia.org/wiki/Bacteriumhttp://en.wikipedia.org/wiki/Mammalianhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Medicationhttp://en.wikipedia.org/wiki/Agricultural


2/48

agents and planting the cell suspension on a glass/plastic substrate, where the cells adhere and

grow. hen cells from primary culture are serially transferred it causes a selection of a single

cell type to become predominant and multiply at a constant rate. he primary cell culture is

then said to have originated a cell strain . -ife span is limited to to 0 passages and then it

dies out and the strain comes to an end. 1uring multiplication of a cell strain some cells

become altered and ac#uire a different morphology, grow faster and have unlimited life. t is

now designated as a cell line.Established cell lines r c ntinu us cell culture! eg 2ep3, 45!67 . +ne in which

the cell nuclei contain chromosome numbers other than the diploid number (heteroploid). he

cells are capable of indefinite replication, not anchorage dependent, not inhibited by density

of population, malignant, and capable of growing in defined media consisting of low

molecular weight nutrients without protein supplements. he transformed cell lines or

modified cell lines are those cells which are either derived from tumor cells or have been

manipulated in some way i.e., transfection with oncogenes or treatment with carcinogens.

"#" Ty$es % mammalian cultures

∗ E&$lant cultures * &ell culture initiated from a fragment of tissue/ organ planted in the

medium/plasma clot. he cells migrate out and form a monolayer, and the original tissue

becomes necrotic.∗ ' n layer cultures! &onsists as a layer of cells growing on the surface of a culture vessel.

-imitation here is the surface area available for the cells to adhere.∗ (us$ensi n Culture! "nchorage!independent cells grow while suspended in fluid medium.

his would yield very large populations of cells.∗ 'icr )carrier cell cultures! 8sed for large scale propagation of anchorage!dependent cells

that cannot be cultivated in suspension, but attached to the surface of carrier particles.

"#* Ad+anta,es % cell culture

$eduction of animal use &ytotoxicity and screening of pharmaceutics, cosmetics,

etc.

$eagent saving $educed volumes, direct access, lower cost

9hysico!chemical environment &ontrolled p2, temperature, osmolarity, dissolved gases

2


3/48

&ell line homogeneity "vailability of selective media, cloning

&haracterization &ytology and immuno!staining are easily performed

9reservation &an be stored in li#uid nitrogen

9hysiological conditions &ontrol of hormone and nutrient concentrations

"#- A$$licati ns % cell culture∗ 4ass culture of animal cell lines is fundamental to the manufacture of viral vaccines and

many biotechnology products including enzymes, hormones, immunobiologicals

(monoclonal antibodies, interleukins, lymphokines) and anticancer agents.∗ 1iagnosis and therapy∗ :creening and studies of cell toxicity mechanisms

CHAPTER 2ANI'AL CELL CULTURE E((ENTIAL( AND LA. 'AINTENANCE

*#" Cell culture lab

"ny laboratory, in which tissue culture techni#ues are performed, regardless of the

specific purpose, must contain a number of basic facilities. hese usually include the

following*

∗ " general washing area (9reparation area)∗ &ulture area

a/ 0ashin, Areahe washing area should contain large sinks, some lead!lined to resist acids and

alkalis, draining boards, and racks, and have access to demineralized water, distilled water,

and double!distilled water. :pace for drying ovens or racks, automated dishwashers, acid

3


4/48

baths, pipette washers and driers, and storage cabinets should also be available in the washing

area.

b/ Culture Areahe most desirable arrangement is a small dust!free room e#uipped with an overhead

ultraviolet light and a positive!pressure ventilation unit. he ventilation should be e#uipped

with a high!efficiency particulate air (259") filter. " .;!


5/48

maintaining cell culture stocks has not changed, the functioning and operation of &+ 3

incubators has become more accurate, more reliable and more convenient.

b/ Laminar %l 1 cabinet

" laminar flow cabinet or laminar flow closet or tissue culture hood is a carefullyenclosed bench designed to prevent contamination of semiconductor wafers, biological

samples, or any particle sensitive device. "ir is drawn through a 259" filter and blown in a

very smooth, laminar flow towards the user. he cabinet is usually

made of stainless steel with no gaps or Aoints where spores might

collect. -aminar flow cabinets may have a 8B!& germicidal lamp to

sterilize the shell and contents when not in use. hese hoods are

generally available in C!ft and 0!ft lengths, the latter being somewhatmore convenient in terms of work space. wo people can work side

by side in a 0!ft hood. his is convenient if the experimental protocol re#uires two people to

work together. -arger hoods also may be necessary if large!scale tissue culture work is to be

done where many large spinners or roller bottles are handled at the same time.c/ In+erted micr sc $e

"n inverted microscope is a microscope with its light source and

condenser on the top, above the stage pointing down, while the obAectives and

turret are below the stage pointing up. nverted microscopes are useful for observing living cells or organisms at the bottom of a large container (e.g., a

tissue culture flask) under more natural conditions than on a glass slide which

is the case with a conventional microscope. he stage on an inverted

microscope is usually fixed, and focus is adAusted by

moving the obAective lens along a vertical axis to bring it closer to or

further from the specimen. he focus mechanism typically has a dual

concentric knob for coarse and fine adAustments. 1epending on the sizeof the microscope, four to six obAective lenses of different

magnifications may be fitted to a rotating turret known as a nosepiece.

hese microscopes may also be fitted with accessories for fitting still!

and video cameras, fluorescence illumination , confocal scanning and many other

applications.

d/ Cry can&ryocans are used for cryopreservation, which is a process where cells or whole

tissues are preserved by cooling to low sub!zero temperatures , such as (typically) >> D or

5

http://en.wikipedia.org/wiki/HEPAhttp://en.wikipedia.org/wiki/Laminar_flowhttp://en.wikipedia.org/wiki/Stainless_steelhttp://en.wikipedia.org/wiki/UV-Chttp://en.wikipedia.org/wiki/Germicidal_lamphttp://en.wikipedia.org/wiki/Microscopehttp://en.wikipedia.org/wiki/Light_sourcehttp://en.wikipedia.org/wiki/Condenserhttp://en.wikipedia.org/wiki/Objective_(optics)http://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Tissue_culturehttp://en.wikipedia.org/wiki/Objective_lenshttp://en.wikipedia.org/wiki/Fluorescence_microscopehttp://en.wikipedia.org/wiki/Confocal_laser_scanning_microscopyhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Biological_tissuehttp://en.wikipedia.org/wiki/Temperaturehttp://en.wikipedia.org/wiki/HEPAhttp://en.wikipedia.org/wiki/Laminar_flowhttp://en.wikipedia.org/wiki/Stainless_steelhttp://en.wikipedia.org/wiki/UV-Chttp://en.wikipedia.org/wiki/Germicidal_lamphttp://en.wikipedia.org/wiki/Microscopehttp://en.wikipedia.org/wiki/Light_sourcehttp://en.wikipedia.org/wiki/Condenserhttp://en.wikipedia.org/wiki/Objective_(optics)http://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Organismhttp://en.wikipedia.org/wiki/Tissue_culturehttp://en.wikipedia.org/wiki/Objective_lenshttp://en.wikipedia.org/wiki/Fluorescence_microscopehttp://en.wikipedia.org/wiki/Confocal_laser_scanning_microscopyhttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Biological_tissuehttp://en.wikipedia.org/wiki/Temperature


6/48

E6=0 F& (the boiling point of li#uid nitrogen ). "t these low temperatures, any biological

activity, including the biochemical reactions that would lead to cell death , is effectively

stopped. 2owever, when cryoprotectant solutions are not used, the cells being preserved are

often damaged due to freezing during the approach to low temperatures or warming to room

temperature. 9henomena which can cause damage to cells during cryopreservation mainly

occur during the freezing stage, and include solution effects, extracellular ice formation,

dehydration and intracellular ice formation. 4any of these effects can be reduced by using

cryoprotectants . 4ost commonly used cryoprotectants are 14:+ and glycerol.

E&$anded e2ui$ment and additi nal su$$lies!

∗ ater bath

∗ &entrifuge

∗ $efrigerator and freezer (!3 F&)

∗ &ell counter (e.g., &ountessG "utomated &ell &ounter or hemocytometer)

∗ :terilizer (i.e., autoclave)

∗ &onfocal microscope

∗ Hlow cytometer

∗ ater de!ionizing unit

∗ Hluorescent microscope

∗ 4icroplate reader

∗ 1eep freezer (!7 I&)

6

http://en.wikipedia.org/wiki/Liquid_nitrogenhttp://en.wikipedia.org/wiki/Cell_deathhttp://en.wikipedia.org/wiki/Cryoprotectanthttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Freezinghttp://en.wikipedia.org/wiki/Extracellularhttp://en.wikipedia.org/wiki/Intracellularhttp://en.wikipedia.org/wiki/Cryoprotectanthttp://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/cell-culture-equipment/basic-laboratory-equipment.htmlhttp://en.wikipedia.org/wiki/Liquid_nitrogenhttp://en.wikipedia.org/wiki/Cell_deathhttp://en.wikipedia.org/wiki/Cryoprotectanthttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Freezinghttp://en.wikipedia.org/wiki/Extracellularhttp://en.wikipedia.org/wiki/Intracellularhttp://en.wikipedia.org/wiki/Cryoprotectanthttp://www.invitrogen.com/site/us/en/home/References/gibco-cell-culture-basics/cell-culture-equipment/basic-laboratory-equipment.html


7/48

∗ 4icropipettes and/or multi!channel pipettes

∗ p2 meter

∗ 4icrogram balance (5lectronic balance)

∗ -ab oven

∗ "J5 and 9"J5 apparatus and accessories

∗ "spiration pump (peristaltic or vacuum)

∗ &ell culture vessels (e.g., flasks, 9etri dishes, roller bottles, multi!well plates)

∗ :yringes and needles

∗ aste containers

*#- Ase$tic techni2ue and , d cell culture $racticehis is to ensure that all cell culture procedures are performed to a standard that will

prevent contamination from bacteria, fungi and mycoplasma, and cross contamination with

other cell lines.∗ :anitize the cabinet using > ? ethanol every time before commencing work.∗ :anitize gloves by wiping them in > ? ethanol and allow them to air dry for ;

seconds before commencing work.∗ 9ut all materials and e#uipment into the cabinet prior to starting work after sanitizing

the exterior surfaces with > ? ethanol.∗ hile working, do not contaminate hands or gloves by touching anything outside the

cabinet (especially face and hair). f gloves become contaminated, re!sanitize with

> ? ethanol as above before proceeding.∗ 1iscard gloves after handling contaminated cultures and at the end of all cell culture

procedures each time.∗ 5#uipment in the cabinet or that which will be taken into the cabinet during cell

culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped

with tissue soaked with > ? ethanol prior to use.∗ 4ovement within and immediately outside the cabinet must not be rapid. :low

movement will allow the air within the cabinet to circulate properly.

7


8/48

∗ :peech, sneezing and coughing must be directed away from the cabinet so as not to

disrupt the airflow.∗ "fter completing work disinfect all e#uipment and material before removing from the

cabinet. :pray the work surfaces inside the cabinet with > ? ethanol and wipe dry

with tissue. 1ispose off tissue by autoclaving.∗ :anitize the cabinet with 6 K ; min 8B light before commencing work and after

finishing work. Warning * 9lastics will crack and become brittle over time with

repeated exposure to 8B light. +nly some cabinets have timed 8B lights. 5nsure they

are not left on for extended periods.

*#3 4umi,ati n % r ms usin, % rmaldehyde$emove all unnecessary items from the room, including those that must not be exposed to the

gas, e.g., cell cultures, sensitive electrical e#uipment, etc.∗ &lean the room with > ? ethanol to minimize the level of microbial contamination

and to allow good gas penetration∗ urn off air!handling system (roof 259" filter system) and close the room as far as

possible and turn on the air conditioner to fan mode.∗ Hill the container with formalin solution (6 ml/m ; of room volume).∗ 9lace the container in middle of the room and leave the room.∗ -ock the door and display safety warning notice.∗ -eave for 3C!>3 h.∗ "fterwards, turn on cabinet and /or air!handling system.∗ -eave until the level of formaldehyde reaches an acceptable level.∗ "lternatively ammonia solution can be placed in a bowl and kept in the room as

ammonia absorbs the formalin vapors and neutralize them.∗ he room may re#uire cleaning to remove residues of paraformaldehyde.

*#5 0ashin, and sterili6ati n % reusable lab)1are∗ :oak in chromic acid solution except metals or directly in detergent.∗ ash with detergent.

∗ $inse with tap!water (continuously or with se#uential changes).∗ $inse with distilled water (two or more times).∗ 1ry in hot air oven.∗ :terilize in autoclave.∗ :tore for use (in dedicated cupboards or racks).

8


9/48

CHAPTER 3PRI'ARY CELL CULTURE 7LY'P8OCYTE CULTURE/

AND 9ARYOTYINGhe culture of explants taken directly from living organism (e.g., biopsy material) is

known as primary cell culture. he culture consists of mixed population of cell types.

Hre#uently, some of the cells survive without undergoing proliferation and will, therefore, be

lost in the increasing population of those which are able to multiply in the conditions supplied

in vitro . &ells from explants may sometimes be converted to cell lines by passage. hese may

continue to proliferate for a number of cell generations. n some instances the primary cells

are fused with so!called immortal (cancer) cells to produce a hybridoma line. 4any of theexplanted cells will survive only for one or a few passages before dying. "part from study of

the primary cells per se there are a number of other uses for these cells including the use of

primary fibroblasts as feeder layers for the growth of some embryonic stem cell types. his

chapter deals with the primary culture of lymphocytes from human blood and osteoblast cells

from rat calvariae.

-#" 0.C is lati n and cultureOutline

-ayer whole citrated blood or plasma, depleted in red cells by dextran!accelerated

sedimentation, on top of a dense layer of Hi-sep . "fter centrifugation, most of the

lymphocytes are found at the interface between the Hi-sep /metrizoate and the plasma.Re2uirements

∗ lood sample in heparin or citrate (concentration determined by collection container

which will already contain citrate or heparin).

∗ &lean centrifuge tubes or universal containers

9


10/48

∗ 9 :

∗ Hi-sep

∗ :erum!free medium

∗ :yringe, 9asteur pipettes or pipette

Pr cedure

∗ &ollect the blood sample in citrated or heparinized container and transport to

laboratory.

NOTE: Human blood may be infected with agents such as I!" virus or hepatitis and

should be handled with great care#

∗ 1ilute 6*3 with 9 :, and layer C ml onto 3 ml Hi-sep . his should be done in a wide,

transparent centrifuge tube with a cap, such as 6 !ml sterile conical tube.

∗ &entrifuge the suspension for 3 !; min at 3 rpm.

∗ &arefully remove the plasma/9 : without disturbing the interface.

∗ &ollect the interface with a syringe or 9asteur pipette, and dilute it to 3 ml in serum!

free medium.

∗ NOTE: $asteur pipettes and syringes with sharp needles should not be used with

human blood# Instead% use a &ml pipetter or a syringe with a blunt canula#

∗ &entrifuge the diluted cell suspension from the interface at 3 rpm for 6 min.

∗ 1iscard the supernatant, and re!suspend the pellet in 3 ml of serum!free medium or

9 :. f several washes are re#uired K e.g., to remove serum factors, re!suspend the

10


11/48

cells in 3 !ml of serum!free medium, and centrifuge two or three times more, and

finally re!suspend the pellet in 3 ml of serum!free medium.

∗ -ymphocytes will be concentrated at the interface, along with some platelets and

monocytes. :ome granulocytes may be found in the interface although most will be

found mostly in the Hi-sep /metrizoate, in the pellet created in step ;. 4onocytes and

residual granulocytes can be removed from the interface fraction by taking advantage

of their adherence to glass (beads or the surface of a flask) or to nylon mesh. 8se a

positive sort by 4"&: or flow cytometry with specific lymphocyte surface markers if

purer preparations are re#uired.

-#* 8ar+est % $eri$heral bl d lym$h cytes % r chr m s me $re$arati nOutline

o obtain metaphase chromosome preparation from peripheral blood following a

modified method of 2ungerford (6=0 ) and to stain the preparation.Re2uirementsColchicine

. 6? colchicine is prepared by dissolving mg of colchicine in ml of double

distilled water. Hypotonic solution

. = g of D&l is dissolved in 6 ml of double distilled water and stored in wash

bottles at ;> o&.Giemsa’s stain

he stock solution of Jiemsa is prepared by dissolving 6g of Jiemsa powder in Cml

of glycerol at 0 o& for 0 to = min using a magnetic stirrer. "fter cooling to room

temperature, 7Cml of methanol is added and left on the magnetic stirrer for one hour. t is

filtered (Lo.6 hatman filter paper) and the filtrate is stored in the refrigerator. he working

solution is prepared by adding 3ml of stock Jiemsa%s solution and 3ml of 6 ? disodium

hydrogen phosphate solution to C0ml of distilled water (p2 0.7).Pr cedure

∗ "t the end of the incubation period, add 6 Ml of . 6? colchicine solution to arrest

the dividing cells at metaphase.∗ ncubate the cultures further for a period of 6 hr. ransfer the contents of the vial to a

centrifuge tube and spin at 6 rpm for min.∗ 1iscard the supernatant and gently tap the cell button. "dd 0ml of hypotonic solution

( . > 4 D&l) and mix gently using a cyclomixer.∗ 9lace the tube in a water bath at ;> o& for 6 min, then add 6 to 3 ml of cold fixative

(methanol * glacial acetic acid , ;*6) using a vortex and spin at 6 rpm for min.

11


12/48

∗ 1iscard the supernatant and re!suspend the cells in freshly prepared fixative. Jive

another change of fixative and leave the material in the refrigerator overnight.∗ :uspend the cell button in a small #uantity of fresh fixative (after the cells have been

spun down) and prepare a test slide by placing a drop of the cell suspension on a

cooled slide and dry over a slide warmer kept at C o&.∗ 5xamine the slide under a light microscope and check the cell density and the spread

of chromosomes.∗ 9repare the rest of the slides after making necessary dilutions of the cell suspension.∗ :tain the slides in a C? buffered solution of Jiemsa%s for to > min, rinse in distilled

water and air!dry. 8nder low (6 x) and high (C x) power obAective lenses of the

microscope, observe several cells in metaphase, with chromosomes showing varying

degrees of condensation amidst interphase nuclei.∗ +bserve Jiemsa%s!stained chromosomes of a metaphase cell (under 6 x obAective)

and count the number of chromosomes.∗ 5ach chromosome will be seen to consist of two sister chromatids held together at the

centromere.∗ 2uman chromosomes are classified into seven groups (" to J) based on gross

morphology i.e., length and position of centromere.

-#- G : .andin, techni2ue usin, try$sin and Giemsa;s 7GTG/Jiemsa is the most popular stain for chromosome analysis. &onventional or solid

staining method is very useful in studying satellites, dicentric chromosomes, ring

chromosomes, fragments, double minutes, fragile sites, and breaks. 2owever, chromosome

identification is not possible.1ifferential staining techni#ues include the fluorescent and Jiemsa%s staining

methods that expound light and dark bands along the length of chromosomes. hese methods

are indispensable in the une#uivocal identification of chromosomes. he Jiemsa bands

obtained by digesting the chromosomes with the proteolytic enzyme, trypsin, are the most

widely used in clinical laboratories for routine chromosome analysis.Outline o band metaphase chromosomes on a given slide employing J!banding techni#ue

using trypsin and Jiemsa%s (J J) (:eabright, 6=>6).Re2uirements

rypsin (2i4edia), C ? buffered Jiemsa solution, sodium chloride (:$-).Trypsin solution

rypsin 7 mg.=? La&l ml

Pr t c l

12


13/48


14/48

Pr t c l&ut around the chromosomes leaving some margin of background. f overlapping is

present, cut out one chromosome and then cut the other from another print. "rrange the

chromosomes on a format based on their banding patterns according to :&L (3 =).

he characteristic banding pattern of each chromosome is listed below*Jroup "&hromosome 6* he short arm has two broad bands over the proximal half and four

bands over the middle and distal part of the long arm. he secondary constriction at the

proximal long arm stains heavily and is polymorphic.&hromosome 3* Hour to seven bands may be present over the entire long arm and three to

four over the short arm. he centromere is only lightly stained.&hromosome ;* he centromere and the paracentric region are fairly densely stained. he

distal parts of both arms stain intensely while the central parts of both arms remain light and

appear as a white band.Jroup &hromosome C* Jenerally more evenly and densely stained. Hour evenly spaced wide bands

are present over the long arm and one or two over the short arm.&hromosome * " heavily stained broad band covers the middle third of the long arm. t is

separated from a telomeric band by absence of staining over the distal part of the long arm.Jroup &&hromosome 0* he short arm has a wide proximal area of rather faint staining which sets

off a heavily stained distal #uarter of the short arms resembling satellites. he paracentric

region of both arms and the entire long arm, except its telomeric region, are heavily stained.&hromosome >* hree distinct bands are found over the long arm. he centromere is

stained. he short arm has two to three bands, one being telomeric.&hromosome 7* " broad band, sometimes split into three to five individual bands, is located

over the middle part of the long arm. ts proximal region stains lightly. he short arm has one

to two bands.&hromosome =* he middle and distal part of the long arm have two wide bands which are

sometimes split into two bands each. he secondary constriction at the proximal long arm

remains light with both Jiemsa%s and #uinacrine. t is highly polymorphic and stains almost

selectively with the Jiemsa!66 techni#ue.&hromosome 6 * he long arm regularly has three bands and the most proximal band stains

most heavily . he short arm has one broad or two narrow bands over the middle part.&hromosome 66* wo bands are located over the middle part of the long arm. hey may be

separated by a narrow negative band. he centromere is stained and separated from a broad

positive band on the short arm by a narrow negative band.&hromosome 63* he OJ%!pattern is similar to chromosome 66 but the central band is over

the proximal part in the long arm and is wider than in chromosome 66.

14


15/48

N chromosome * here are two to four well marked bands over the long arm, the proximal

one being most prominent, and one over the middle part of the short arm. Lo difference exists

between the N chromosomes in female individuals.Jroup 1

&hromosome 6;* he long arm stains positively over most of its length except for a narrowtelomeric segment. 8p to four individual bands may be present.&hromosome 6C* " broad proximally located band, which may be split into three bands, and

a negatively stained band over the distal third characterize the long arm. here is a narrow

distal band of medium intensity at the end of the long arm.&hromosome 6 * he distal half of the long arm is lightly stained. he proximal half of this

arm has two to three bands.Jroup 5&hromosome 60* he paracentric secondary constriction at the proximal arm is heavily

stained in contrast to its negative fluorescence. t is highly variable as a polymorphic trait.

here is a lightly stained band over the Aunction of the distal to the middle third of the long

arm. he short arm generally shows light staining, but one to two bands may be present.&hromosome6>* he entire chromosome takes little stain and remains rather pale. here is

one usually narrow band over the short arm and one over the distal long arm.&hromosome 67* his is a densely stained chromosome. wo broad bands appear over the

long arm, the proximal one being brighter and broader than the other. " median narrow band

may be present over the short arm.Jroup H&hromosome 6=* here is a fairly positively stained area around the centromere.&hromosome 3 * he distal ends of both arms stain positively while the centromere remains

less stained than in chromosome 6=.Jroup J&hromosome 36* t is darker and smaller than &hr 33. n particular, the long arm is densely

stained with one to two proximal bands discernible.&hromosome 33* here is stain mainly around the centromere, with a pale band in the center

of the long arm.P chromosome* he distal half of the long arm is the most densely stained region of the

karyotype with #uinacrine. t is variable in length and polymorphic. wo bands may be

visible, the proximal one generally being darker. he short arm and the proximal long arm are

usually positively stained.

15


16/48

CHAPTER 4'AINTANENCE AND CULTURE O4 E(TA.LI(8ED CELL LINE(he maAority of the cells are derived from vertebrates. ith the exception of

hematopoietic cell lines and a few others, most are anchorage!dependent and have to be

cultured on a suitable substrate that is specifically treated to allow cell adhesion and

spreading (i.e., tissue!culture treated / . 2owever, many cell lines can also be adapted for

suspension culture. &ells that are cultured in suspension can be maintained in culture flasks

that are not tissue!culture treated, but as the culture volume to surface area is increased

beyond which ade#uate gas exchange is hindered (usually .3 K . m-/cm 3), the medium

re#uires agitation. his agitation is usually achieved with a magnetic stirrer or rotating

spinner flasks. he difference between adherent and suspension cell cultures and their

advantages/disadvantages are as follows*

Adherent Cell Culture (us$ensi n Cell Culture

"ppropriate for most cell types, including

primary cultures

"ppropriate for cells adapted to suspension

culture and a few other cell lines that are non!

adhesive (e.g., hematopoietic)

$e#uires periodic passaging, but allows easy

visual inspection under inverted microscope

5asier to passage, but re#uires daily cell

counts and viability determination to follow

growth patterns@ culture can be diluted to

stimulate growth

&ells are dissociated enzymatically (e.g.,

ryp-5Q 5xpress, trypsin) or mechanically

1oes not re#uire enzymatic or mechanical

dissociation

Jrowth is limited by surface area, which may

limit product yields

Jrowth is limited by concentration of cells in

the medium, which allows easy scale!up

Jrowth is limited by concentration of cells in

the medium, which allows easy scale!up

&an be maintained in culture vessels that are

not tissue!culture treated, but re#uires

agitation (i.e., shaking or stirring) for

ade#uate gas exchange

8sed for cytology, harvesting products 8sed for bulk protein production, batch

16


17/48

continuously, and many research applications harvesting, and many research applications

3#" Cell line Re$ sit ries!he culture may be established from primary cells, or established cell cultures may be

procured from commercial or non!profit suppliers (i.e., cell banks). $eputed suppliers

provide high #uality cell lines that are carefully tested for their integrity and to ensure that the

culture is free from contaminants. $egardless of their source, it is to be made sure that all

new cell lines are tested for mycoplasma contamination before one begins to use them. he

mission of cell repositories focuses on the ac#uisition, authentication, production,

preservation, development and distribution of standard reference microorganisms, and cell

lines for research in the life sciences. he maAor cell repositories are as follows*

" && ("merican type &ulture &ollection)5&"&& (5uropean &ollection of "nimal &ell &ultures)1:4R (1eutsche :ammlung von 4ikroorganismen und Rellkulturen ! Jerman

&ollection of 4icroorganisms and &ell &ultures)S&$ (Sapanese &ollection of $esearch ioresources)

&ell line $epository in ndia*he repository at Lational &entre for &ell :cience (L&&: complex, 8niversity of

9une &ampus, Janeshkhind, 9une C66 >, 4aharashtra, ndia. ebsite*

http*//www.nccs.res.in/ , 5mail* patoleTnccs.res.in , milindpatoleThotmail.com) is the only

repository that houses human and animal cells in ndia. he L&&: repository serves toreceive, identify, maintain, store, cultivate and supply animal and human cell lines and

hybridomas. he repository has procured cultures from various sources within the country

and abroad from ; animal species. " maAor bulk of the cell lines stocked in the repository

has been procured from the "merican ype &ulture &ollection (" &&) and the 5uropean

&ollection of "nimal &ell &ultures (5&"&&)."fter receiving cells from organizations such as " && or L&&:, the flask in which

the cells are received should be wiped thoroughly with > ? ethanol and the morphology of

the cells should be observed. he cells should also be checked for contamination or any

change in the medium. "fter clear observation, the cells are kept in &+ 3 incubator for one

day without any disturbance. he next day, the medium is removed and the cells are sub!

cultured for further processing.

3#" (ubculture % Adherent 7m n layer/ Cell Lines

17

http://www.nccs.res.in/mailto:[email protected]://www.nccs.res.in/mailto:[email protected]


18/48

"dherent cell lines will grow in vitro until they have covered the surface area

available or the medium is depleted of nutrients. 9rior to this point the cell lines should be

sub!cultured in order to prevent the culture dying. o subculture the cells they need to be

brought into suspension. he degree of adhesion varies from cell line to cell line but in

maAority of cases proteases, e.g. trypsin, is used to release the cells from the flask.∗ 5yeball the cells ! Biew cultures using an inverted microscope to assess the degree of

confluency and confirm the absence of bacterial and fungal contaminants.∗ &heck the p2 of the culture medium by looking at the color of the indicator, phenol

red. "s a culture becomes more acid the indicator shifts from red to yellow!red to

yellow. "s the culture becomes more alkaline the color shifts from red to fuchsia (red

with a purple tinge). "s a generalization, cells can tolerate slight acidity better than

they can tolerate shifts in p2 above p2 >.0.∗ &ell attachment* "re most of the cells well attached and spread outU "re the floating

cells dividing cells or dying cells which may have an irregular appearanceU∗ 9ercent confluency* he growth of a culture can be estimated by following it toward

the development of a full cell sheet (confluent culture). y comparing the amount of

space covered by cells with the unoccupied spaces one can estimate percent

confluency.∗ &ell shape is an important guide* $ound cells in a un!crowded culture is not a good

sign unless these happen to be dividing cells. -ook for doublets or dividing cells. Jet

to know the effect of crowding on cell shape.∗ -ook for giant cells* he number of giant cells will increase as a culture ages or

declines in well!being. he fre#uency of giant cells should be relatively low and

constant under uniform culture conditions.∗ +ne of the most valuable guides in assessing the success of a culture split is the rate

at which the cells in the newly established cultures attach and spread out. "ttachment

within an hour or two suggests that the cells have not been traumatized and that the in

vitro environment is not grossly abnormal. -onger attachment times are suggestive of

problems. Levertheless, good cultures may result even if attachment does not occur

for four hours.∗ Deep in mind that some cells will show oriented growth patterns under some

circumstances while many transformed cells, because of a lack of contact inhibition

may pile up especially when the culture becomes crowded. Jet to recognize the

range of cells shapes and growth patterns exhibited by each cell line.

Pr cedure % r sub)culturin, % cells!

18


19/48

∗ $emove the spent medium.∗ ash the cell monolayer with 6!3 ml of 9 : without &a 3V/4g 3V (&4H!9 :).∗ 9ipette trypsin onto the washed cell monolayer using 6ml per 3 cm 3 of surface area.

(e.g., 6 ml K 3 , ; ml > ). $otate flask to cover the monolayer with trypsin.

∗ ncubate in hood for 3!6 minutes depending on the cell line. :ome cells may need to be sitting in the incubator. oo long of a period of trypsinisation, the cells will die.

Lot enough, you will not transfer enough cells.∗ +nce the cells start to sheet and the media becomes cloudy, move on to the next step.

Pou may examine the cells using an inverted microscope to ensure that many (WC ?)

the cells are detached and floating. t may help to Xslap or tapY the flasks gently to

release any remaining attached cells.∗ $e!suspend the cells in a small volume of fresh serum!containing medium to

inactivate the trypsin. 1isperse the cells (this is a process to disaggregate clumps or

sheets of cells) by running the suspended cells in the medium three to four times with

the tip of the pipette on the bottom corner of the flask. e careful not to aspirate your

media into the pipette aid. f this happens, the filter must be replaced.∗ ransfer the re#uired number of cells to a new labeled flask containing pre!warmed

medium.9ey P ints

∗ rypsin is inactivated in the presence of serum. herefore, it is essential to remove all

traces of serum from the culture medium by washing the monolayer of cells with 9 :

without &a 3V/4g 3V∗ &ells should only be exposed to trypsin/51 " long enough to detach cells. 9rolonged

exposure could damage surface receptors.∗ rypsin should be neutralized with serum prior to seeding cells into new flasks,

otherwise cells will not attach.

(ub)culture % sus$ensi n cells!:ub!culturing suspension cells is somewhat less complicated than passaging adherent

cells. ecause the cells are already suspended in growth medium, there is no need to treat

them enzymatically to detach them from the surface of the culture vessel, and the whole

process is faster and less traumatic for the cells. $eplacement of growth medium is not

carried out in suspension cultures@ instead, the cells are maintained by feeding them every 3

to ; days until they reach confluency. his can be done by directly diluting the cells in the

culture flask and continue expanding them, or by withdrawing a portion of the cells from the

19


20/48

culture flask and diluting the remaining cells down to a seeding density appropriate for the

cell line.

3#* Cell c untin, and +iability assay8em cyt meter cell c unt 7Try$an blue stainin,/

∗ horoughly clean the hemocytometer and its cover slip and wipe both with > ?

alcohol before use.∗ 9lace the cover slip centrally over the counting area and across the grooves. Jently

move the cover slip back and forth over the chamber to appropriate position on the

ruler area.∗ 4ix the cell suspension gently and add an ali#uot to the trypan blue solution (6 µ l

cell suspension* 6 µ l dye). he dilution will depend on the cell concentration.∗ 1raw a sample into a micro!pipette after mixing thoroughly and the entire tip on the

pipette to rest at the Aunction between the counting chamber and the cover slip.∗ 8sing a light microscope at low power, focus on the counting chamber.∗ &ount the viable and non!viable cells in both halves of the chamber.

Calculati ns!otal number of viable cells Z " x x & x 6 C

otal dead cell count Z " x x 1 x 6 C

otal cell count Z viable cell count V dead cell count? Biability Z Biable cell count x 6 / otal cell count

here " Z Bolume of cells Z dilution factor in trypan blue

&Z mean number of unstained cells1Z mean number of dead or stained cells6 C is the conversion factor for .6mm ; to 6 4

3#- Cry $reser+ati nf a cell line can be expanded sufficiently, preservation of cells by freezing will allow

secure stocks to be maintained without aging and protect them from problems of

contamination, incubator failure, or medium and serum crises. deally, 6 [ 6 0 [ 6 > cells

should be frozen in 6 ampoules, but smaller stocks can be used if a surplus is not available.

20


21/48

he normal procedure is to freeze a stock of one to three ampoules as soon as surplus cells

are available, then to expand remaining cultures to confirm the identity of the cells and

absence of contamination, and freeze down a seed stock of 6 K3 ampoules. +ne ampoule,

thawed from this stock, can then be used to generate a using stock. n many cases, there may

not be sufficient doublings available to expand the stock as much as this, but it is worth

saving some as frozen stock, no matter how little, although survival will tend to decrease

below 6 [ 6 0 cells/ml and may not be possible below 6 [ 6 cells/ml.Hactors favoring good survival after freezing and thawing are*

(i) 2igh cell density at freezing (6 [ 6 0 [ 6 > cells/ml).(ii) 9resence of a preservative, such as glycerol or dimethyl sulfoxide (14:+) at K

6 ?.(iii):low cooling, 6 \&/min, down to E> \& and then rapid transfer to a li#uid nitrogen

freezer.(iv) $apid thawing.(v) :low dilution, ∼3 !fold, in medium to dilute out the preservative.(vi) $e!seeding at 3! to !fold the normal seeding concentration. Hor example, if cells

are frozen at [ 6 0 cells in 6 ml of freezing medium with 6 ? 14:+ and then

thawed and diluted 6*3 , the cell concentration will still be 3 # [ 6 cells/ml at

seeding, higher than the normal seeding concentration for most cell lines, and the

14:+ concentration will be reduced to . ?, which most cells will tolerate for 3C h.(vii) &hanging medium the following day (or as soon as all the cells have attached) to

remove the cryoprotectant. here cells are more sensitive to the cryoprotectant, they

may be centrifuged after slow dilution and re!suspended in fresh medium, but this

step should be avoided if possible as centrifugation itself may be damaging to freshly

thawed cells.

9 r cedure!

∗ +nce cells get 7 != ? confluent, the cells are washed with 9 : and trypsinized.

∗ 6x6 0!6x6 > cells/ml is taken along with 6 ? serum medium and cryo!protectant such

as dimethyl sulfoxide (14:+* !6 ?) in a cryovial.

∗ hen the cells are kept for slow cooling, 6F&/min, down to !> F& and then the vials

are rapidly transferred to a li#uid nitrogen freezer.

3#3 Resuscitati n % 4r 6en Cell Lines

21


22/48


23/48

∗ &entrifuge the suspension to pellet the cells. $e!suspend the cells in growth medium,

and count them.

∗ 1ilute the cells depending on the growth rate of the cell line and allowing 6 !3 ml of

cell suspension per =0 well plate.

∗ ransfer the cell suspension to a boat and, with a pipette (multichannel pipette is

preferred), add 3 µ l of the suspension into each well of the central 6 columns of a

=0!well plate, starting with column 3 and ending with column 63 and placing . K 6

x 6 ; cells into each well.

∗ "dd 3 µ l of growth medium to the eight wells in columns 6. &olumn 6 will be usedto blank the plate reader.

∗ ncubate the plates in a humidified atmosphere at ;> ο& for 3C hrs, such that the cells

are in the exponential phase of growth at the time the drug / toxicant is added.

Dru, ? t &ins additi n∗ 9repare a serial five!fold dilution of the cytotoxic drug / compound in growth

medium to give ten concentrations. his set of concentrations should be chosen

such that the highest concentration kills most of the cells and the lowest kills none

of the cells. +nce the cytotoxicity of a drug is known, a smaller range of

concentrations can be used.

=>)1ell $late ma$! 6 3 ; C 0 > 7 = 6 66 63

lank

&ontrol 6 3 ; C 0 > 7 = 6

23


24/48

∗ Z lank (4edia only)

∗ & Z &ontrol (8ntreated cells)

∗ 6! 6 Z 1ose range of drug/toxin

Pr cedure!∗ $emove the growth medium using pipette. ake care that the tip of the pipette does

not touch the cell sheet.∗ "dd re#uired volume of growth medium to the cell sheet and add medium containing

defined concentrations of the drug.∗ Hollow the same for solvent control.∗ "dd 3 µ l of the medium free from the drug to the control wells.∗ ncubate the plates at ;> ο& in ? &+ 3 environment for re#uired time points.

5#" 'TT AssayPrinci$le

4 (;!(C, !1imethylthiazol!3!yl)!3, !diphenyltetrazolium bromide) is cleaved by

mitochondrial dehydrogenase of viable cells, yielding a measurable purple formazan product.

his formazan production is proportionate to the viable cell number and inversely

proportional to the degree of cytotoxicity. Hormazan can be #uantified

spectrophotometrically. his assay measures the antiproliferative effects of cytotoxic drugs.

Pr cedure ∗ "dd 3 µ l of mg/ml of 4 to each well.∗ rap the plates in aluminium foil and incubate in dark for 3 to C h at ;> ο &.∗ $emove the medium and 4 from the wells and dissolve the remaining 4 !

formazan crystals by adding 6 µ l of 14:+ to all the wells.∗ $ecord absorbance in a micro!plate reader at > nm (measurement) and 0; nm

(reference) immediately, since the product is unstable.

24


25/48

Determinati n % IC 5@he half maximal inhibitory concentration ( & ) is a measure of the effectiveness of a

compound in inhibiting biological or biochemical function. his #uantitative measure

indicates how much of a particular drug or other substance ( inhibitor ) is needed to inhibit a

given biological process (or component of a process, i.e. an enzyme , cell , cell receptor or

microorganism ) by half. n other words, it is the half maximal ( ?) inhibitory concentration

( &) of a substance ( ? &, or & ). t is commonly used as a measure of antagonist drug

potency in pharmacological research.

'eth d!∗ 9lot a graph of the absorbance (y!axis) against the concentration of the drug (x!axis).∗ he & concentration is determined as the drug concentration that is re#uired to

reduce the absorbance to half that of the control.∗ he absolute value of the absorbance should be plotted so that control values may be

compared, but the data can then be converted to a percentage!inhibition curve, to

normalize a series of curves.

∗ he percentage inhibition is calculated, from the data, using the formula*

4ean +1 of untreated cells (control) ! 4ean +1 of treated cells

4ean +1 of untreated cells (control)

∗ he graph can be plotted with percentage of inhibition (y!axis) against the

concentration of drug (x!axis) and & concentration is determined.

25

x 6=

http://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_receptorhttp://en.wikipedia.org/wiki/Microorganismhttp://en.wikipedia.org/wiki/Receptor_antagonistshttp://en.wikipedia.org/wiki/Potency_(pharmacology)http://en.wikipedia.org/wiki/Enzyme_inhibitorhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_receptorhttp://en.wikipedia.org/wiki/Microorganismhttp://en.wikipedia.org/wiki/Receptor_antagonistshttp://en.wikipedia.org/wiki/Potency_(pharmacology)


26/48

CHAPTER 6'ORP8OLOGICAL AND OT8ER 'ICRO(COPIC 'ET8OD( 4OR A((E(('ENT

O4 CELL DEAT8

here are different modes of cell death caused due to toxicity and this chapter deals

with the various morphological and other microscopic methods for assessment of cell death

' r$h l ,ical assessment % the cells>#" Acridine Oran,e?Ethidium .r mide 7AO?E./ stainin,!Princi$le!

"cridine orange is a vital dye and will stain both live and dead cells. 5thidium

bromide will stain only cells that have lost membrane integrity, i.e., 5 will permeate only

cells which have lost membrane integrity. -ive cells will appear uniformly green. 5arly

apoptotic cells will stain

green and contain bright

green dots in the nuclei

as a conse#uence of

chromatin condensation and

nuclear fragmentation.

-ate apoptotic cells will

also incorporate ethidium bromide and therefore stain orange but, in contrast to necrotic cells,

the late apoptotic cells will show condensed and often fragmented nuclei. Lecrotic cells stain

orange, but have a nuclear morphology resembling that of viable cells, with no condensed

chromatin.

26


27/48

Fig: Photomicrographs of AO/EB stained HEp-2cells incubated for 24 hrs A! untreated controlcells" B! to#in treated cells$ arro%s sho%ing

(tain Pre$arati n(t c

Dru, ? t &ins additi n!)

reat the cells with & concentration of the cytotoxic drug / compound prepared in

growth medium. +ne of the wells should be left without drug as control.

ncubate the cells according to experimental set up (for e.g., 3C or C7 hrs).

(tainin,!)

∗ oth the live and dead cells should be collected for the morphological studies. hesupernatant containing the dead cells are first collected and centrifuged (C rpm for

C mins) in an eppendorf. hen the remaining live cells adhering to the well surface are

trypsinized, neutralized, centrifuged and collected in the same eppendorf. $esuspend

the cells with growth medium.∗ 4ix the cell suspension with the "+/5 stain in 6*6 ratio on a microscopic slide and

cover with glass cover slip.∗ 5xamine at least ; cells in a fluorescent microscope in C x obAective using

fluorescein filter ( 450–490 nm).

27


28/48


29/48




(tainin,!)

∗ oth the live and dead cells should be collected for the morphological studies. he

supernatant containing the dead cells are first collected and centrifuged (C rpm for



the cells with growth medium.∗ 4ix the cell suspension with the 2oechst ;;3 7 stain in 6*6 ratio on a microscopic

slide and cover with glass cover slip.∗ 5xamine at least ; cells in a fluorescent microscope in C x obAective using

fluorescein filter ( 377–355 nm).

>#- BC)" (tainin,Princi$le

4itochondrial membrane potential, ]^m, is an important parameter of mitochondrial

function used as an indicator of cell health. S&!6 is a lipophilic, cationic dye that canselectively enter the mitochondria and reversibly change color from green to red as the

membrane potential increases. n healthy cells with high mitochondrial ]^m, S&!6

spontaneously forms complexes known as S!aggregates with intense red fluorescence. +n the

other hand, in apoptotic or unhealthy cells with low ]^m, S&!6 remains in the monomeric

form, which shows only green fluorescence.

29


30/48

(tain $re$arati n!(t c

Pr cedure!)(eedin,!)

∗ rypsinize a sub!confluent monolayer culture, and neutralize the cells with growth

medium containing serum.∗ &entrifuge and collect the pellet. $e!suspend the cells in growth medium and count

the cells using hemocytometer.∗ 9ut a cover slip inside each well of the 0 well plate then add ; ml of growth medium

and seed around 6, , cells per well in the 0 well plate.


reat the cells present in each well of the 0 well plate (with cover slip) with &concentration of the cytotoxic drug / compound prepared in growth medium. +ne of the wells

should be left without drug as control.


Pr cedure

∗ "fter the completion of drug treatment period incubate the cells present in the

wells which has glass cover slips with the S& 6 working solution at ;>I& for 3

minutes.∗ "fter 3 min, take out the cover slip with the adhered cells and keep it on the glass

slide inversely.∗ he directly observe the mitochondrial depolarization patterns of treated and

control

cells in

a fluorescent microscope fitted with a ;>>!; nm filter, at C x magnification

and then take photographs.

30

Fig: Photomicrographs of ,)- stained .iHa cer ical cancer cells for 0 h" A! control cells$ B! treated cells


31/48

>#3 Anne&in : Cy- Assay % r A$ $t sisPrinci$le!

he annexins are a group of homologous proteins that bind phospholipids in the

presence of calcium. n living cells phosphatidylserine _9:` is transported to the inner plasma

membrane leaflet by the enzyme 4g!" 9 dependent aminophospho!lipid translocase.

2owever, during the onset of apoptosis, 9: is transported to the external leaflet of the plasma

membrane. 9: is then available for binding to annexin B and any of its conAugates in the

presence of &a 3V ions.

"poptotic cells can be differentiated from necrotic cells in several ways*∗ "nnexin!&y;.67 ("nn!&y;) binds to phosphatidylserine present in the outer

leaflet of the plasma membrane of cells starting the apoptotic process. he binding

is observed as red fluorescence.∗ 0!&arboxyfluorescein diacetate (0!&H1") is used to measure viability. hen this

non!fluorescent compound enters living cells, esterases present hydrolyze it,

producing the fluorescent compound, 0!carboxyfluorescein (0!&H). his appears

as green fluorescence.

∗ &ells can be incubated either with "nn!&y; or 0!&H1" separately, or with the twocompounds simultaneously. "fter labeling at room temperature, the cells are

immediately observed by fluorescent microscopy. -ive cells will be labeled only

with 0!&H (green), while necrotic cells will be labeled only with "nn!&y; (red).

&ells in the early stage of apoptosis, however, will be labeled with both &y; (red)

and 0!&H (green).

(eedin,!)

∗ rypsinize a sub!confluent monolayer culture, and neutralize the cells with growthmedium containing serum.

∗ &entrifuge and collect the pellet. $e!suspend the cells in growth medium and count

the cells using hemocytometer.∗ "dd ; ml of growth media in each well and seed around 6, , cells per well in the

0 well plate.


31


32/48




(tainin,!)

∗ oth the live and dead cells should be collected for the morphological studies. he

supernatant containing the dead cells are first collected and centrifuged (C rpm for



the cells with growth medium.∗ 4ix the cell suspension with the "nnexin stain in 6*6 ratio on a microscopic slide and

cover with glass cover slip.∗ 5xamine at least ; cells in a fluorescent microscope in C x obAective using

fluorescein filter.

>#5 C met assay % r DNA dama,ehe comet assay or single cell gel electrophoresis (:&J5) is a rapid, sensitive and

relatively simple method for detecting 1L" damage at the level of individual cells.

Rea,ents

6) 6 ? Lormal agarose

3) 6 ? -ow melting agarose;) 5lectrophoresis buffer (p2 6 )

32

Fig: 1 A fragmentation in to#in treated .iHa cer ical cancer cells as re ealed in the comet assa+" )omet images of 1 Adouble strand brea3s at 2 and 24 h treatment of to#in"


33/48


34/48

Pre$arati n % slides

∗ 1rop gently the 3 Ml of 6? normal agarose in 9 : at 0 I& on to a fully frosted

micro!slide, cover immediately with a cover slip and place over a frozen ice pack

for about min.

∗ $emove the cover slip after the gel had set. 4ix the cell suspension from one

fraction with 6? low melting agarose at ;>I& in 6*; ratio.

∗ "pply 6 Ml of this mixture #uickly on top of the gel, coated over the micro!slide

and allow to solidify as before.

∗ Jive a third coating of 6 Ml of 6? low melting agarose on the gel containing the

cell suspension and allow to solidify.

∗ :imilarly, prepare the slides for each cell fraction.

Cell lysis

∗ "fter solidification of the agarose, remove the cover slips and immerse the slides

in ice!cold lysis solution at CI& for 60h.

∗ 9erform all the above operations in low lighting conditions in order to avoid any

additional 1L" damage.

Electr $h resis

∗ 9lace the slides horizontally in an electrophoresis tank after being removing them

from the lysis solution.

∗ Hill the reservoirs with electrophoresis buffer until the slides Aust immerse in it.

∗ "llow the slides to stand in the buffer for about 3 min (to allow 1L" unwinding)

after which carry out the electrophoresis at .7v/cm for 6 min.

34


35/48

∗ "fter electrophoresis, remove the slides, wash thrice in neutralization buffer and

gently dab to dry.

∗ "dd a few drops of the working solution of ethidium bromide on to the gel and

cover the slide with a cover slip.

∗ 5xamine the stained 1L" in the cells at 3 x and C x magnifications using a

fluorescent microscope e#uipped with a ;0 nm excitation filter and a C; nm

barrier filter.

∗ 4easure the lengths of 1L" migration (comet tail) in these cells using &":9

software. :core about 0 !6 comets per point.

A$$licati n % CA(P s %t1are

he comets are analysed using &":9 software. he images are used to estimate the

1L" content of individual nuclei and to evaluate the degree of 1L" damage representing the

fraction of total 1L" in the tail. &ells are assigned to five classes* ( >? of the 1L" in the

tail undamaged), 6 (>K6 ?), 3 (6 K33?), ; (33K; ?) and C ( ; ?, maximally damaged)accordingly.

CHAPTER 7'OLECULAR TEC8NI UE( 4OR A((E((ING TOXICITY IN VITRO

35


36/48

he development of molecular techni#ues has yielded innovative alternative tools for

understanding and demonstrating the mechanisms underlying various biological functions. t

has also shed considerable light on a number of targeted action mechanisms of various

pharmaceutical, toxicological agents and provides a guide for researchers to study the

molecular basis of the various effects caused by these agents. his chapter deals with a few

molecular techni#ues for assessing toxicity in vitro#

#" Immun cyt chemistry 7ICC/

a/ Per &idase c nFu,ated (u$er (ensiti+e P lymerH meth d % r cell culturesIntr ducti n

&& is the techni#ue used to detect and localize antigens by means of labeled

antibodies through "g!"b interaction that are visualized by enzyme!substrate!chromogen

reaction.Princi$le

n these techni#ues an enzyme!labeled antibody is used to link a cellular antigen

specifically to a chromogen that can be more readily visualized in light microscope.A l t % meth ds are a+ailable in this techni2uei/ Direct labelin, meth d

n this method, the enzymes are directly labeled with the primary "b, so it is a direct

method. his method is more expensive and less in sensitivity.ii/ Indirect labelin, meth d

n this method, the enzymes are labeled with the 3I"b, which was produced against primary "b. :o it is an indirect method. his method is very cheap and easy to handle.iii/ A.C meth d

n this method, the 3I"b and avidin are labeled with the biotin and enzyme,

respectively. "vidin has high affinity to biotin. hese two biomolecules are used to elongate

the chain and make possible to detect an earlier stage of cancer.i+/ P lymeric meth d

his techni#ue permits binding of a large number of enzyme molecules to a secondary

antibody via the dextran backbone. he benefits are many, including increased sensitivity,minimized non!specific background staining and a reduction in the total number of assay

steps as compared to conventional techni#ue.he simple protocol is∗ "pplication of primary "b∗ "pplication of super enhancer∗ "pplication of enzyme!labeled polymer secondary "b∗ "pplication of the substrate K chromogen.

he result is outstanding sensitivity, signal intensity, low back!ground staining and reduced

non K specific binding.

Aim

36


37/48

o detect and localize cl!3 in cells by means of super sensitive polymer 2$9.o detect and localize p ; in cells by means of super sensitive polymer 2$9.

Re2uired rea,entsI/ Primary Ab;s

6. cl!33. p ;

II/ (u$er sensiti+e $ lymer 8RP . 5lectronic weighing balance7. ; ? 2ydrogen peroxide=. "cetone

6 . 4ethanolRe2uired chemicalsa) 1isodium 51 "

b) risodium citratec) 1isodium hydrogen phosphated) 9otassium dihydrogen phosphatee) :odium chloridef) ris bufferg) 6L 2&lh) 2ematoxylin

Re2uired s luti ns t be $re$ared"@ PLL 7"@@mlJ (i,ma/

t is mainly for used as an adhesive, to keep the cells intact on the slide during

immunostaining.Alternati+es3? "95: in acetone (6 ml@ :igma)&hrome alum Jelatin solutionAR s luti n 7% r A, retrie+al/

ris 51 " buffer* p2! =.ris ! 0. g

1isodium 51 " ! .>CCg1istilled water ! 6 ml

37


38/48

Alternati+es!Citrate bu%%er 7$8) >#@/

risodium citrate ! 3.=C g6L 2&l ! ml1istilled water ! 6 ml

' di%ied citrate bu%%er 7 $8) >#*/&itric "cid (anhydrous) ! 6.=3g1isodium 51 " ! .>CCg1istilled water ! 6 ml

hese solutions are mainly used to unmask the antigen and also to enhance the

reaction between "g "b.

-/ 0ash .u%%ers!Ph s$hate .u%%ered (aline 7P.(/K $8) #3

1isodium hydrogen phosphate ! 6>. g

9otassium dihydrogen phosphate ! 6.:odium chloride ! 6>g1istilled water ! 6 ml

Alternati+es !Tris .u%%er (aline 7T.(/K $8) #3) #>

ris buffer ! .0 g:odium chloride ! 7g6L 2&l ! Cml1istilled water ! 6 ml

hese solutions are mainly used to wash the unbound "b%s from the cells, and also

used to maintain the p2 of the cells.

Pr cedurei) &ell cultures are grown onto "95: ("mino 9ropylene 5thoxy :ilane) coated slides.

Lote*• 1o not allow cells to dry at any stage of the staining procedure.• &arry out the steps of incubation with antibody at ;>I&.• 8se appropriate controls for each antibody tested.

ii) &old acetone ! 6 min K Hixationiii) . ? 2 3+ 3 in 4ethanol ! 6 min K o block endogenous enzymeiv) ransfer to : buffer (p2 >.0) K min x ; times K washingv) 9ressure cookerize in : buffer (p2 K >.0) ! 6 min ! 2eat induced X"gY retrieval.vi) 9lunge the pressure cooker into sink with water ! 3 min ! &ooled to room temperaturevii) ransfer to : buffer (p2 >.0) ! min x ; times K washingviii) 9ower block K 6 to 6 min ! o block non!specific reaction

with the other tissue "g

x) : buffer min x ; times to wash unbound "b%s o enhance the reaction between

primary and secondary "b%sxii) : buffer ! min x ; times K to wash unbound "b%s

o elongate chain andalso to label the enzyme

38

ix) 1rain and cover cells with

concerned primary "b6 hour o identify r markers by "g K "b reaction

xi) :uper!enhancer

xiii) :uper!sensitive poly K 2$9 ! ; min

! ; min


39/48

xiv) : buffer ! min x ; times K to wash unbound "b%sxv) &olour development with working

colour development solutionxvi) : buffer ! min x ; times K o washxvii) ap water K min ! o wash

xviii) 2ematoxylin K 6 min ! o counter!stainxix) ap water K min ! o wash excess stainxx) "ir dry and mount with the mounting medium and view under the microscope.xxi) $esults*

r. 4arkers ! rown Lucleus ! lue

b/ Immun cyt chemistry n cyt l ,y smearsRe2uired materials

6) &old acetone3) . ? 2 3+ 3 in methanol (only for hemopoietic smears);) ris buffer C) 9rimary antibodies

) :uper!sensitive polymer/2$9/1" kitPr cedure

6) "fter smear preparation, allow it to air dry for minimum 67h.3) Hix in cold acetone for 3!; min.;) Hurther fix in . ? 2 3+ 3 in methanol for min ( only for hematopoietic smears)C) Deep in ris buffer for min x ; times

) 9ower block for 6 min0) 1rain and wipe the surrounding of the smears@ incubate with concerned primary

antibody for 6h.>) ash with ris buffer for min x ; times.7) ncubate with super!enhancer for ; min.=) ash with ris buffer for min x ; times.6 ) ncubate with super!sensitive polymer/2$9 for ; min.66) ash with ris buffer for min x ; times.63) 1evelop color with 1" ! working solutions for 7!6 min.6;) ash with ris buffer for min x ; times.6C) ash in distilled water for min.6 ) &ounter!stain with hematoxylin for 6min.60) ash in distilled water for min.6>) "ir dry, clear in xylene, mount with 19N.

Result r. 4arker K rown Luclei ! lue

#* Gene E&$ressi n (tudies Ad $tin, P lymerase Chain Reacti n 7PCR/Intr ducti n

he starting template for a 9&$ reaction can be 1L" or $L". 1L" is usually the

appropriate template for studying the genome of the cell or tissue (as in inherited genetic

diseases, somatic mutation in a tumor, or somatic rearrangement in lymphocytes) and for the

39

!7 min ! o give colour to the "g%s


40/48

detection of 1L" viruses. Hor information on gene expression in a cell or tissue or the

presence of genomic $L" in a retrovirus such as 2 B, $L" is the appropriate template. $L"

can be better than genomic 1L" for detecting structural changes in long genes, since

amplifying the spliced $L" transcript instead of the genomic se#uence greatly reduces the

length of 1L" to be handled without losing any of the coding regions where clinically

significant deletions may be expected.

$ !9&$ combines c1L" synthesis from $L" templates with 9&$ to provide a

rapid, sensitive method for analyzing gene expression. $ !9&$ is used to detect or #uantify

the expression of m$L", often from a small concentration of target $L". he template for

$ !9&$ can be total $L" or poly (") V selected $L". $ reactions can be primed with

random primers, oligo (d ), or a gene!specific primer (J:9) using a reverse transcriptase.

$ !9&$ can be carried out either in two!step or one!step formats. n two!step $ !

9&$, each step is performed under optimal conditions. c1L" synthesis is performed first in

$ buffer and one tenth of the reaction is removed for 9&$. n one!step $ !9&$, reverse

transcription and 9&$ take place se#uentially in a single tube under conditions optimized for

both $ and 9&$.

One (te$ RT)PCR % r *5 l reacti n + lume

his protocol serves as a guideline for one!step $ !9&$ with a 3


41/48

$L") should be included in every experiment

;. 4ix the master mix thoroughly, and dispense a ppropria te volumes into 9&$

tubes. 4ix gently, for example, by pipetting the master mix up and down a few tim e s.

C. "dd template $L" ( e 3


42/48

1enaturation . !6min at = I&.

"nnealing . !6min at ! 7I& ( I& below T m of pri m e r s)

5xtension 6min >3I&

Lumber of cycles 3 !CHinal extension 6 min

Analysis

"nalyze the 9&$ reaction products by agarose gel electrophoresis of a Ml ali#uot

from the total reaction. he products should be readily visible by 8B trans!illumination of the

ethidium bromide!stained gel.

#- Pr tein E&$ressi n (tudies Ad $tin, 0estern .l tPrinci$le

he estern blot is an analytical techni#ue used to detect specific proteins in a given

sample of tissue homogenate or extract. t uses :1: gel electrophoresis to separate proteins

based on their size. he proteins are then transferred to a membrane (typically nitrocellulose

or 9B1H ), where they are detected using antibodies specific to the target protein.E&tracti n % $r teinRea,entsDi,nam bu%%er 7 p2* > to =) (3 ml)

∗ 2epes 6 m4 ( =. > mg)

∗ 4g&l 3 6. m4 (>C.3 Ml from 6 mg/ml stock)

∗ D&l 6 m4 (670.C Ml from 6 mg/ml stock)

∗ 1 . m4 (;7. 0 Ml from mg/ml stock)

∗ 94:H . m4 (C;. C Ml from mg/ml stock)

∗ L9C .6? (3 Ml)

∗ :tore at C &

Lote* 94:H and L9C should be added fresh to the lysis buffer.Pr cedure

42

http://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/Antibodyhttp://en.wikipedia.org/wiki/Analytical_techniquehttp://en.wikipedia.org/wiki/Proteinshttp://en.wikipedia.org/wiki/Gel_electrophoresishttp://en.wikipedia.org/wiki/Nitrocellulosehttp://en.wikipedia.org/wiki/PVDFhttp://en.wikipedia.org/wiki/Antibody


43/48

∗ he cells are collected and centrifuged.

∗ he supernatant is discarded and dignam lysis buffer is added to the pellet.

∗ he pellet is lysed by passing through a fine syringe and incubated in deep freezer

(; hours to overnight, freeze!thaw cycle can also be done).

∗ hen it is thawed and centrifuged at 63, to 6 , rpm at C o &.

∗ he supernatant is collected and stored in deep freezer (!> o &) for later use.

Estimati n % $r tein c ncentrati n 7.rad% rd assay/Princi$le

radford reagent uses &oomassie blue J!3 . ithout protein, the solution is red!

brown and it is an acidic solution. hen protein binds the pDa of the dye shifts causing the

dye to become blue. he dye is measured at = nm in a spectrophotometer. radford dye is

easy to use, fast and sensitive.

Rea,ents

∗ radford reagent

1issolve 6 mg of &oomassie blue J3 in ml of ethanol, add 6 ml of

7 ? of phosphoric acid and make volume to 6 liter with distilled water.

∗ .64 9 :

∗ :tandard protein solution

1issolve mg of :" in ml of .64 9+ C. his solution contains 6

Mg/pm/mlPr cedure

∗ ake .6 ml of sample solution and make the volume to 6 ml with .6 4 9 : (p2

>. ).

∗ 9ipette appropriate ali#uots of :" containing !6 Mg protein. 4ake the

volume to 6ml with .6 4 phosphate buffer (p2 >. ) in all tubes.

43


44/48

∗ "dd ml of radford reagent to all the tubes and mix thoroughly.

∗ $ecord the absorbance at = nm against the reagent blank.

∗ 9lot a standard " = Bs Mg protein in standard.

∗ 1etermine the protein content in the sample extract from the standard curve.

∗ &alculate the amount of protein per ml sample.

P ly)Acrylamide Gel Electr $h resis 7PAGE/ and 0estern .l tRea,ents

"# "crylamide/ is (; ? , 3.0>? &)3=.3 g acrylamide and 7 mg L%L%Kbis!methylene!acrylamide are dissolved in 6

ml of de!ionized water. Hilter and store at C & in the dark (; days maximum).*# 6 ? (w/v) :1:

6 g :1: is dissolved in = ml water with gentle stirring and brought to6 ml with

de!ionized water.-# 6. 4 ris K 2&l (p2 7.7)

67.6 g ris base is dissolved in 7 ml de!ionized water. "dAust p2 to 7.7 with 0 L

2&l and bring total volume to 6 ml with de!ionized water and store at C &.3# . 4 ris K 2&l (p2 0.7)

0 g ris base is dissolved in 0 ml de!ionized water. "dAust p2 to 0.7 with 0 L 2&l

and bring total volume to 6 ml with de!ionized water and store at C &.5# 6 ? "9: (Hresh daily)

6 mg "mmonium persulfate dissolved in 6 ml of de!ionized water.># L% L% ! etramethyl ethylene diamine ( 5451)

&ommercially available# :ample buffer (:1: reducing buffer)

6.3 ml . 4 ris!2&l (p2 0.7), 3. ml glycerol, and 3 ml 6 ? (w/v) :1: are

added to .3 ml of . ? (w/v) bromophenol blue and brought to total volume of =. ml

with ;. ml de!ionized water. he buffer is stored at room temperature.


45/48

6 ? "9: ! .6 ml5451 ! . C ml

? stacking gel is prepared by mixing the reagents as given below.1e!ionized 2 3+ K .> ml; ? degased acrylamide/bis K 6.> ml

. 4 ris K 2&l (p2 0.7) K 3. ml6 ? (w/v) :1: K .6 ml6 ? "9: ! . ml

5451 ! . ml(e$arati n % $r teins

9roteins are separated by :1: K 9olyacrylamide gel electrophoresis based on their

size as described by -aemmli (6=> ). y heating the sample under denaturing and reducing

conditions, proteins become unfolded and coated with :1: detergent molecule, ac#uiring a

high net negative charge that is proportional to the length of the polypeptide chain. hen

loaded on to a gel matrix and placed in an electric field (6 B), the negatively charged

protein molecules migrate towards the positively charged electrode and are separated by a

molecular sieving effect. " molecular weight protein marker ( !galactosidase, ovalbumin,

carbonic anhydrase, !lactoglobulin or lysozyme) that produces band of known size is used to

help identify proteins of interest.Trans%er % $r teins

"fter the separation of proteins by :1:!9"J5, the separating gel is rinsed with

distilled water and e#uilibrated in cold transfer buffer. ransfer sandwich is assembled in the

following order from anode (V) to cathode (!).

Rea,ents

T 1bin bu%%er 7% r trans%er/

Hor 3 ml

ater =;.> ml

ris (3 m4) .>0g

Jlycine ;.0 g

45


46/48

4ethanol ml

:1: . =Cg

p2 7.3!7.C, store at C c

T.(T 7% r 1ashin,/

Hor ml preparation

ris .0g

Lacl C.;7;g

weeen 3 .3 ml

p2 >.C

P.(T 7% r 1ashin,/

9 : ml

weeen 3 .3 ml

p2 >.C

(ubstrate bu%%er

ris 6 6.C3mg/3 ml

" 6 mg

8 *O *

Pr t c l

:oak the gel membrane and pads in towbin for 3 !; min

ransfer at 3 B (constant voltage) for 6hour

locking! 3 hrs at room temperature ( ?milk powder in :)

"dd primary antibody (6*; dilution) and incubate for 3 hrs at $ /or overnight at C c

ash with 9 : (0 times, min each)

ncubate in 3 "b (6*; dilution) in 6hour at room temperature

46


47/48

ash with : (0 times, min each)

"dd substrate

"dd 2 3+ 3 (7 ml)

he set!up is assembled in the following way*

∗ 9ositive end

∗ Hiber pad

∗ Hilter paper soaked in transfer buffer

∗ L& 4embrane

∗ Jel

∗ Hilter paper soaked in transfer buffer

∗ Hiber pad

∗ Legative end

he set up is placed in the transfer apparatus filled with cold transfer buffer and

subAected to electric field..l c


48/48

"fter the unbound probes are washed away, the estern blot is ready for detection of

the probes that are labeled and bound to the protein of interest.

In vitro cell culture technique protocol manual

Documents

Transcript of In vitro cell culture technique protocol manual