Practical 2

Cell cycle

Mitosis

Presentation

Confocal microscopy

Confocal

microscope

Cell cycle

interphase

M

Cell cycle

G1G2

S

M

Cell cycle

S

G2

G1

M

G0 - phase

Checkpoints of the cell cycle

Written test

• 6 minutes

• Put down your name, group and version of

the test.

• Don’t write anything on the question sheet!

• More than one alternative could be

correct.

Task 1: Cell cycle phases, „DNA-ploidy“ examination

• The graph shows numerical distribution of bone marrow cells related to the DNA concentration.

• The cells were stained with a fluorescent dye that binds to DNA.

• Using the fluorescence activated cell sorter (FACS) the cells were divided according to their different DNA concentration in the nuclei.

Task 1 – questions• Why cells of different

DNA concentration are present in the sample?

• Why the curve has two peaks?

• Is it possible to determine relative duration of the cell cycle phases?

Task 1 – result

S

0

nu

mb

er o

f ce

lls

G1

G2 + M

x xDNA content1x 2x

Task 1 - conclusion• Second peak – G2 +

M cells – their the DNA concentration is twice higher than this in G1 cells (first peak).

• Area between two peaks – S phase cells (they have different DNA concentration, therefore they don't create any peak).

S

0

nu

mb

er o

f ce

lls

G1

G2 + M

DNA content1x 2x

Aneuploidy in Barrett's esophagus

Aneuploid cells

Barrett’s esophagus = precancerosis

Mitosis

• Nuclear division

– Prophase

– (Prometaphase)

– Metaphase

– Anaphase

– Telophase

• Cell division

– cytokinesis

Task 2: Observing mouse chromosomes

• Slides were prepared from the bone marrow of mouse.

• The bone marrow was removed from femur of sacrificed mouse and fixed with the mixture of methanol + glacial acetic acid (3:1).

• The suspension of cells was dropped onto the slide and stained with the Giemsa-Romanowski solution (similarly to the human chromosomes in previous practical).

Task 2: How to observe mouse chromosomes

• Find the chromosomes or interphase nuclei on the slide using 6x or 10x objective lens.

• Change the objective magnification into 40 or 45x and observe chromosomes. Compare the picture in the microscope with adjacent photo.

• Enumerate chromosomes in the photo.• How many chromosomes are present in the

nuclei of mouse somatic cells? • Describe their shape and determine type of

chromosomes.

Mouse chromosomes

Task 3: Observing mitosis in onion root tip cells

• The root tip is prepared for staining by treating it with 1M hydrochloric acid, which exposes aldehydes, present in the chromosomal DNA, to which the stain may then attach.

• The root tip is stained with Schiff reagent, a dye mixture containing fuchsine that adheres to the aldehyde groups exposed in step 1, thereby coloring the chromosomes and interphase nuclei a deep magenta.

• The cells are dispersed into a single layer on a slide, and the slide is prepared for microscopic observation.

Principal of Feulgen’s nuclear reaction.

• This reaction is a histochemical evidence of the DNA in the nuclei or chromosomes

Scheme of the Feulgen reaction

Task 3: How to observe mitosis in onion root tips?

• Find cells on the slide using 6x or 10x objective lens.

• Change the objective magnification into 40x or 45x. Find the area where actively dividing cells are present.

• Observe the cells in this area and compare them to adjacent photos until you can easily recognize cells in interphase, prophase, metaphase, anaphase and telophase.

Mitosis in onion root tips

Task 3 – examination of mitotic cells

• Using the same high magnification, count the number of cells in each of these phases: prophase, metaphase, anaphase and telophase.

• Examine approximately 20-30 of dividing cells. Put down their numbers to the table.

• Are portions of cells representing each mitotic phase equal or not? Explain your results.

A table of cells in various mitotic phases

Phase Number Total number

Prophase

Metaphase

Anaphase

Telophase

12

4

6

3

Results and conclusions of

the task 2 and 3

Mouse chromosomes

40 chromosomes – mostly telocentric ones

Mouse karyotype - female

Importance of mouse

chromosomes examination –

mutagenicity (genotoxicity)

screening

Mutagenic factor

• = genotoxic factor

• A chemical, physical or biological agent causing

the DNA damage that could result in mutation.

• Majority of mutagenic agents have carcinogenic

activity (they cause transformation of a normal

cell into a tumor one).

Chromosomal changes in two mouse cells after application of genotoxic agent.

tetraploidy chromosomal breakage

Task 3 - mitosis

• Number of mitotic phases

– The share of cells in various mitotic phases is

different.

– Majority of cells is present in prophase - this

phase takes the longest time period of the M-

phase.

Mitosis in tumor cellsImpairment of cell cycle regulation could result in the

malignant transformation of normal cell. Tumor cells have usually high mitotic activity that leads to chromosomal

defects (mainly changes of chromosome number).

Next practical

• Meiosis, gametogenesis

• Assisted reproduction

• Presentation: Ethical issues of assisted reproduction

• Test – microscopes, cell cycle, mitosis, DNA structure

and its replication

Switch off the microscope lamps and return slides to

boxes, please.

See you next week!

Special staining of mouse chromosomes using such called in situ hybridization method.

Fluorescence Activated Cell Sorter

• The quantitative measurement of the DNA content of cells was one of the earliest applications in flow cytometry. It has become possible by specific fluorescent dyes which bound to the double helix of nuclear DNA. The fluorescence inside the cell nucleus can easily be detected using this method. Thus, the amount of DNA for cell populations can be studied regarding:

– the cell cycle– the ploidy level

• Fluorescence activated cell sorter (FACS) measures DNA concentration according to fluorescence intensity. It allows us to measure DNA concentration in cells of patients with hematological and other malignancies.

back to task 1

Prophase

back

Plant cell Animal cell

Metaphase

back

Plant cell Animal cell

Anaphase

back

Plant cell Animal cell

Telophase

back

Plant cell

Animal cell

Cytokinesis

back

cytokinesis of tumor cells