Bd Lsrii User's Guide

7/30/2019 Bd Lsrii User's Guide

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% t

r a n s m

i s s i o n

longpass


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Appendix A: Technical Overview 97

Shortpass Filters

An SP filter has the opposite properties of a longpass filter. An SP filter passeslight with a shorter wavelength than the filter rating.

Bandpass Filters

A BP filter transmits a relatively narrow range or band of light. Bandpass filtersare typically designated by two numbers. The first number indicates the centerwavelength and the second refers to the width of the band of light that is passed.For example, a 500/50 BP filter transmits light that is centered at 500 nm and has

wavelength (nm)

wavelength (nm)

% t

r a n s m

i s s i o n

shortpass

a total bandwidth of 50 nm. Therefore, this filter transmits light between 475and 525 nm.

Figure A-5 Bandpass filter

bandpass


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98 BD LSR II Users Guide

BP and DF filters have the same general functionthey transmit a relativelynarrow band of light. The principal difference between them is theirconstruction. DF filters have more cavities or layers of optical coatings, resultingin a steeper transmission curve than the curve for a BP filter. This steep slopemeans that a DF filter is better at blocking light outside the rated bandwidth of the filter.

wavelength (nm)

% t r

a n s m

i s s i o n

t r a n s m

i s s i o n

BP 500/50 filter

DF 500/50 filter

AF 500/50 filter


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Dichroic Mirrors

Dichroic filters that are used to direct different color light signals to differentdetectors are called dichroic mirrors or beam splitters.

Although dichroic mirrors have the properties of LP or SP optical filters, you cannot necessarily use any type of LP or SP filter as a beam splitter. A beam splittermust have a surface coating that reflects certain wavelengths, but many LP or SPfilters are absorbance filters that do not have any specific reflectivecharacteristics. Also, optical filters and beam splitters are rated at a specific angleof incidence. When used in front of the fluorescence detectors, they areperpendicular to the incident light, and when used as a beam splitter, they areplaced at an angle relative to the light source. Their optical properties aretherefore designed for that angle of incidence.

Compensation Theory

Fluorochromes emit light over a range of wavelengths ( Figure A-3 on page 95 ).Optical filters are used to limit the range of frequencies measured by a givendetector. However, when two or more fluorochromes are used, the overlap inwavelength ranges often makes it impossible for optical filters to isolate lightfrom a given fluorochrome. As a result, light emitted from one fluorochrome

wavelength (nm)

% t

appears in a detector intended for another ( Figure A-6 ). This is referred to asspillover. Spillover can be corrected mathematically by using a method calledcompensation.

Figure A-6 Spillover from the FITC fluorochrome to the PE detector


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For example, FITC emission appears primarily in the FITC detector, but some of its fluorescence spills over into the PE detector. The spillover is corrected orcompensated forhence the term fluorescence compensation.

Figure A-6 shows that some of the FITC emission appears in the PE detector.This can be seen in a dot plot of FITC vs PE.

Figure A-7 Theoretical display of FITC vs PE without compensation

n o r m a l

i z e d

i n t e n s

i t y

PE

FITC

unstainedparticles

FITC-positive

population

This FITC spillover in the PE detector is to be corrected as indicated by the arrowin Figure A-7 . Using the Compensation tab of the Cytometer window inBD FACSDiva software, you can adjust the PE-%FITC spectral overlap value.Compensation is optimal when the positive and negative FITC populations have

the same means or medians in the PE parameter statistics.Figure A-8 FITC spillover optimally compensated out of the PE parameter


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Once fluorescence compensation has been set for any sample, the compensationsetting remains valid for a subsequent dim or bright sample, because

compensation subtracts a percentage of the fluorescence intensity. Figure A-9 illustrates this principle. Although the signals differ in intensity, the percentage of the FITC spillover into the PE detector remains constant.

PE

FITC

matching means

unstainedparticles

FITC-positivepopulation

Figure A-9 Two FITC signals of different intensity

FITC PE

different intensity FITC signals

y


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Electronics

As cells or other particles pass through a focused laser beam, they scatter thelaser light and can emit fluorescence. Because the laser beam is focused on a small

spot and particles move rapidly through the flow cell, the scatter or fluorescenceemission has a very brief durationonly a few microseconds. This brief flash of light is converted into an electrical signal by the detectors. The electrical signal iscalled a pulse.

1 A pulse begins when a particle enters the laser beam. At this point, both thebeam intensity and signal intensity are low.

2 The pulse reaches a maximum intensity or height when the particle reachesthe middle of the beam, where the beam and signal intensity are thebrightest. The peak intensity, or height of the pulse, is measured at thispoint.

3 As the particle leaves the beam, the pulse trails off below the threshold.

same proportion or percentage of spectral overlap in PE channel n o

r m a

l i z e

d i n t e n s i t y

Figure A-10 Anatomy of a pulse

it y

s i g n a l

i n t e n s

i t y

time


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time

s i g n a

l i n t e n s

i ty

s i g n a l

i n t e n s i t y

time

Pulse Measurements

The pulse processors measure pulses by three characteristics: height, area, andwidth.

Figure A-11 Pulse measurements


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Pulse height is the maximum digitized intensity measured for the pulse.

Pulse area is an integration of the digitized measures over time.

Pulse width calculates:

height

threshold

baseline

0 volts

window gate:width

voltage

time

area

areaheight x 64,000

Digital Electronics

BD LSR II flow cytometer electronics digitize the signal intensity produced by adetector. The digitized data is stored in memory and further processed by theelectronics to calculate

Pulse height, area, and width

C ti


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Compensation

Parameter ratios

These results are transferred to your workstation computer for further processingby BD FACSDiva software. For more information about digital theory, refer toDigital Theory in the BD FACSDiva Software Reference Manual .

Threshold

The threshold is the level at which the system starts to measure signal pulses.A threshold is defined for a specific detector signal. The system continuouslysamples the digitized signal data and calculates pulse area, height, and width forall channels based on the time interval during which the threshold is exceeded.

Thresholds can also be set for more than one parameter, and pulse measures arebased on either of the following:

Intervals during which ALL signals exceed their threshold value

Intervals during which ANY signal exceeds its threshold value

Laser Controls

Controls in the Laser tab of the Cytometer window are used to manually set the(laser) delay, area scaling, and window extension values.

These parameters are set by BD Biosciences service personnel during theBD LSR II flow cytometer installation and performance check and are updatedeach time you run a performance check.


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If needed, see Optimizing Laser Delay on page 157 for instructions on manuallyadjusting laser delay settings. Do not otherwise change the settings in the Laser

tab unless instructed to do so by BD Biosciences. Changing the settings affectsyour data.

Appendix B


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107

Troubleshooting

The tips in this section are designed to help you troubleshoot your experiments.You can find additional troubleshooting information in the BD FACSDivaSoftware Reference Manual .

If additional assistance is required, contact your local BD Biosciences technical

support representative. See Technical Assistance on page xv.

Cytometer Troubleshooting

Observation Possible Causes Recommended Solutions

Droplet containmentvacuum not functioning

Worn O-ring in retainer Replace the O-ring. See Changingthe Sample Tube O-Ring onpage 90 .

O t l i t t d i 1 L th t i ( Fig 4 4


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Outer sleeve is not seated inthe retainer

1 Loosen the retainer ( Figure 4-4on page 88 ).

2 Push the outer sleeve up intothe retainer until seated.

3 Tighten the retainer.

Outer sleeve is not on thesample injection tube

Replace the outer sleeve.

1 Loosen the retainer.

2 Slide the outer sleeve over thesample injection tube until it isseated.

3 Tighten the retainer.

Waste line is pinched,preventing proper aspiration

Check the waste line.

Waste tank is full Empty the waste tank.

Sample tube not fittingon SIP Sample tube other thanBD Falcon tubes used Use BD Falcon 12 x 75-mmsample tubes. See Equipment onpage 120.

Worn Bal seal Replace the Bal seal. See Changingthe Bal Seal on page 88.

Rapid sample aspiration Support arm is to the side Place the support arm under thesample tube.

Droplet containment moduleis failing

Call your service representative.

No events in acquisitiondisplay and RUN buttonis green.

Threshold is not set to thecorrect parameter (usuallyFSC)

Set the threshold to the correctparameter for your application.

Threshold level is too high Lower the threshold level.

Cytometer Troubleshooting (continued)



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Appendix B: Troubleshooting 109

PMT voltage for thresholdparameter is set too low

Set the PMT voltage higher for thethreshold parameter.

Gating issue Refer to the BD FACSDivaSoftware Reference Manual forinformation on setting gates.

Air in the sheath filter Purge the filter. See Removing AirBubbles on page 49.

No sample in the tube Add sample to the tube or install anew sample tube.

Sample is not mixed properly Mix the sample to suspend cells.

Waste tank is full Empty the waste tank.

PMT voltages set too low ortoo high for displayparameter

Reset the PMT voltages.

Too few events are displayed Increase the number of events todisplay.

Sample injection tube isclogged

Remove the sample tube to allowbackflushing.

If the event rate is still erratic,clean the sample injection tube.See Daily Cleaning and Shutdownon page 78.

Bal seal is worn Replace the Bal seal. See Changingthe Bal Seal on page 88.

No events in acquisitiondisplay and RUN buttonis green (continued)

Laser is not warmed up Wait the recommended amount of time for the laser to warm up.

30 min for the 488-nm (blue)

30 min for the 355-nm (UV)




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15 min for the 405-nm (violet)

20 min for the 633-nm (red)

Laser delay is set incorrectly Adjust the laser delay settings.

See Setting Laser Delay onpage 155 .

Laser is not functioning Verify the malfunction bychanging the threshold to analternative laser while running theappropriate sample. If unsuccessful, contactBD Biosciences.

No events in acquisitiondisplay and RUN buttonis orange

RUN is not activated Press the RUN button.

Sample tube is not installedor is not properly seated

Install the sample tube correctlyon the SIP.

Sample tube is cracked Replace the sample tube




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Sample tube is cracked Replace the sample tube.

Sheath container is not

pressurized

Ensure that the sheath

container lid and all connectorsare securely seated.

Inspect the O-ring and replaceit if necessary. See Changing theSample Tube O-Ring onpage 90.

Bal seal is worn Replace the Bal seal. See Changing

the Bal Seal on page 88.Air leak at sheath container Ensure that the sheath container

lid and all connectors are securelyseated.

No events in acquisitiondisplay and RUN buttonis orange (continued)

Sheath container is empty Fill the sheath container.

Air in sheath filter Purge the filter. See Removing Air

Bubbles on page 49.

No fluorescent signal Incorrect fluorochromeassignment

Make sure the cytometerconfiguration in the softwarematches the optical filters in thecytometer.

Wrong filter is installed Make sure the appropriate filter is




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Wrong filter is installed Make sure the appropriate filter isinstalled for each fluorochrome.See Changing Optical Filters orMirrors on page 43.

Laser is not functioning Verify the laser malfunction bychanging the threshold to analternative laser while running theappropriate sample. If unsuccessful, contactBD Biosciences.

High event rate Air bubble in the sheath filteror flow cell

Remove the air bubble. SeeRemoving Air Bubbles onpage 49.

Threshold level is too low Increase the threshold level. Referto the BD FACSDiva SoftwareReference Manual forinstructions.

PMT voltage for thethreshold parameter set toohigh

Set the PMT voltage lower for thethreshold parameter. Refer to theBD FACSDiva SoftwareReference Manual forinstructions.

Sample is too concentrated Dilute the sample.

Sample flow rate is set on HI Set the sample flow rate to MEDor LO.

Low event rate Threshold level is too high Lower the threshold level. Refer tothe BD FACSDiva SoftwareReference Manual forinstructions.

PMT voltage for the Set the PMT voltage higher for the




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PMT voltage for thethreshold parameter is set toolow

Set the PMT voltage higher for thethreshold parameter. Refer to theBD FACSDiva SoftwareReference Manual forinstructions.

Sample is not adequatelymixed

Mix the sample to suspend thecells.

Sample is too diluted Concentrate the sample. If theflow rate setting is not critical tothe application, set the flow rateswitch to MED or HI.



If the event rate is still erratic,clean the sample injection tube.See Daily Cleaning and Shutdown

on page 78.Erratic event rate Sample tube is cracked Replace the sample tube.

Bal seal is worn Replace the Bal seal. See Changingthe Bal Seal on page 88.



If the event rate is still erratic,clean the sample injection tube.See Daily Cleaning and Shutdownon page 78.

Erratic event rate(continued)



If the event rate is still erratic,clean the sample injection tube.See Daily Cleaning and Shutdown


http://-/?-http://-/?-


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on page 78.

Contaminated sample Prepare the specimen again.Ensure that the tube is clean.

Sheath filter is dirty Replace the fil ter. See Changingthe Shea th Filter on page 85.

Distorted scatterparameters

Cytometer settings areimproperly adjusted

Optimize the scatter parameters.Refer to the BD FACSDivaSoftware Reference Manual forinstructions.

Air bubble in sheath filter orflow cell

Purge the air from the filter. SeeRemoving Air Bubbles onpage 49.

Flow cell is dirty Perform the system flushprocedure. See System Flush onpage 80.

Air leak at sheath container Ensure that the sheath containerlid is tight and all connectors aresecure.

Hypertonic buffers or fixative Replace the buffers and fixative.

Excessive amount of debris in display

Threshold level is too low Increase the threshold level.

Sheath filter is dirty Replace the filter. See Changingthe Sheath Filter on page 85.

Flow cell is dirty Flush the system. See System Flush


http://-/?-http://-/?-http://-/?-http://-/?-


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on page 80.

Dead cells or debris in sample Examine the sample under amicroscope.

Sample is contaminated Re-stain the sample, ensure tube isclean.

Stock sheath fluid iscontaminated

Rinse the sheath container with DIwater, then fill with sheath fluidfrom another (or new lot) bulk

container.High CV Air bubble in sheath filter or

flow cellPurge the filter. See Removing AirBubbles on page 49.

Sample flow rate is set toohigh

Set the sample flow rate lower.

Air leak at sheath container Ensure that the sheath containerlid is tight and all connectors aresecure.

Flow cell is dirty Flush the system. See System Flushon page 80.

Poor sample preparation Repeat sample preparation.

Sample not diluted in samefluid as sheath fluid

Dilute the sample in the same fluidas you are using for sheath.

Poor QC results Air bubble or debris in flowcell

Prime the fluidics system. SeePriming the Fluidics on page 50.

Old or contaminated QCparticles

Make new QC samples andperform the quality controlprocedure again.




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Sample not diluted in same

fluid as sheath fluid

Dilute the sample in the same fluid

as you are using for sheath.Laser not warmed up Wait the recommended amount of

time for the laser to warm up.

30 min for the 488-nm (blue)

30 min for the 355-nm (UV)

15 min for the 405-nm (violet)

20 min for the 633-nm (red)

Laser not functioning Contact BD Biosciences.

Optical alignment problem Contact BD Biosciences.

Appendix C

Supplies and Consumables


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117

Supplies and Consumables

To order spare parts and consumables, such as bulk fluids, from BD Biosciences:

Within the US, call (877) 232-8995.

Outside the US, contact your local BD Biosciences customer support

representative.

Worldwide contact information can be found at bdbiosciences.com.

Use the following part numbers to order supplies for your BD LSR II system:

QC Particles on page 118

QC Cytometer Setup and Tracking Particles on page 118

Reagents on page 119

Equipment on page 120

QC Particles

Particle Laser Supplier Catalog No.

SPHERORainbowCalibration Particles(8 peak)

SPHERO UltraR i b

all

all

BD Biosciences

Spherotech, Inc.

559123

URFP-30 2


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QC Cytometer Setup and Tracking Particles

RainbowFluorescent Particles

(single peak)

30-2

DNA QC Particles kit blue 488 nm BD Biosciences 349523

Particle Laser Supplier

BD Cytometer Setupand Tracking beads

UV (355 nm and 375 nm)

violet (405 nm and 407 nm)

blue (488 nm)

green (532 nm)

red (633 nm and 645 nm)

BD Biosciences(contact BDBiosciences for moreinformation)

Reagents

Reagent Supplier Catalog No.

BD FACSFlow sheath fluid BD Biosciences 340398(US and Latin

America)

342003(Europe)


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Appendix C: Supplies and Consumables 119

Monoclonal antibodies BD Biosciences a

a. Refer to the BD Biosciences Product Catalog or the BD Biosciences website (bdbiosciences.com).

BD FACS lysing solution BD Biosciences 349202

BD FACSRinse solution BD Biosciences 340346

BD FACSClean solution BD Biosciences 340345

Dyes and fluorochromes BD Biosciences

Molecular Probes

Sigma

Chlorine bleach (5% sodiumhypochlorite)

Clorox or other majorsupplier (to ensure thatthe bleach is at thecorrect concentrationand free of particulatematter)

Equipment

Equipment Item Supplier Catalog No.

Bal seal BD Biosciences 343509

O-ring, sample tube 343615

Sheath filter assembly 344678

BD Falcon TM polystyrene test 352052


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p y ytubes, 12 x 75-mm 352054

352058

Appendix D

Standard Base Configuration


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121

g

The standard base configuration for a BD LSR II cytometer supports detectors,filters, and mirrors for one to four lasers. This appendix describes how to set upthe cytometer optics using standard default configuration components.

4-Blue 2-Violet 2-355 UV 2-Red Configuration on page 122

Additional Optics on page 128

The BD LSR II cytometer can also be ordered with one of several optionalconfigurations, which are described in Appendix E .

4-Blue 2-Violet 2-355 UV 2-Red Configuration

The standard configuration supports a blue octagon, and violet, UV, and redtrigons. Table D-1 shows the detectors, filters, and mirrors used in the standarddefault configuration, and recommended fluorochromes for each detector. Theword blank indicates that a blank optical holder should be used instead of amirror or filter. A dash () indicates that no slot exists for a mirror in that PMTposition.

Table D-1 Default filters and fluorochromes


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Detector Array(Laser)

PMT(Detector)

LongpassDichroicMirror

BandpassFilter

Fluorochrome orScatter Parameter

blue octagon(488-nm laser)

A 735 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 550 LP 575/26 PE, PID 505 LP 530/30 FITC, GFP

E blank 488/10 SSC

F blank blank none

G blank blank none

H blank none

violet trigon(405-nm laser)

A 505 LP 525/50 AmCyan

B blank 440/40 Pacific Blue

C blank none

UV trigon(355-nm laser)

A 505 LP 530/30 Indo-1 (Blue)

B blank 450/50 Indo-1 (Violet), DAPI

C blank none

red trigon(633-nm laser)

A 735 LP 780/60 APC-Cy7

B blank 660/20 APC

C blank none

Table D-1 Default filters and fluorochromes (continued)


PMT(Detector)


BandpassFilter



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Appendix D: Standard Base Configuration 123

Base ConfigurationFigure D-1 shows a default base cytometer configuration.

Figure D-1 Base configuration


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Octagon and Trigon MapsThis section shows how to install mirrors and filters in your octagon and trigonsfor the standard default configuration.

If a slot is filled with a filter or mirror, an identifying number appears in thatposition on the configuration map. If a slot is filled with a blank optical holder,

that position on the configuration map is unlabeled.

Figure D-2 Standard default configuration: blue octagon

F I TC

6 9 5 / 4 0

P e r C P -C y 5.5

488-nm blue laser


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4 8 8 /

1 0

5 3 0 / 3 0

5 0 5 L P

T C

S S C

6 8 5 LP

7 8 0 / 6 0

7 3 5 L P

5 7 5 / 2 6

5 5 0 L P

P E - C y 7 P E

Figure D-3 Standard default configuration: red and violet trigons

6 6 0 / 2 0

AP C

4 4 0 / 4 0

P ac i f i c Bl ue

633-nm red laser 405-nm violet laser


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7 8 0 / 6 0

7 3 5 L P

A P C - C y 7

5 2 5 / 5 0

5 0 5 L P

A m C y a n

Figure D-4 Standard default configuration: UV trigon

4 5 0 / 5 0

D AP I

355-nm UV laser


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5 3 0 / 3 0

5 0 5 L P

I n d o - 1 ( B l u e )

Additional OpticsThis section describes some common custom filter and mirror configurations.Table D-2 shows the detector arrays, mirrors, and filters used in the customconfigurations, and recommended fluorochromes for each detector. The mirrorsand filters used in these custom configurations are contained in the BD LSR IIcytometer spares kit.

Table D-2 Additional filters and mirrors

( ) l l h


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Maps on the following pages show how to install mirrors and filters in youroctagon and trigons for common custom configurations.

Detector Array (Laser) Mirror Filter Fluorochrome

blue octagonstandard 488-nm blue laser

600 LP 610/20 PE-Texas Red

635 LP 670/14 PerCPBD Cy-Chrome reagent

585/42 DsRed

violet trigonoptional 405-nm violet laser

none none none

UV trigonoptional 355-nm UV laser

450 LP Indo-1 (Blue)

405/20 Indo-1 (Violet)

red trigonoptional 633-nm red laser

none none none

PE-Texas Red

To use PE-Texas Red, replace the mirror and filter for the B PMT of the blueoctagon as shown below.

488-nm blue laser


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4 8 8 /

1 0

5 3 0 / 3 0 5 0 5 L P

F I T C

S S C

6 1 0 / 2 0

6 0 0 LP

7 8 0 / 6 0

7 3 5 L P

5 7 5 / 2 6

5 5 0 L P

P E - C y 7 P E

P E -T e x as Re d

Indo-1

If you have a violet laser, replace the mirror for the A PMT of the UV trigon asshown on the left below.

If you do not have a violet laser, replace both the mirror for the A PMT and thefilter for the B PMT of the UV trigon as shown on the right below.

355-nm UV laser(with violet laser)

355-nm UV laser(no violet laser)


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4 5 0 / 5 0

5 3 0 / 3 0

4 5 0 L P

I n d o - 1 ( B l u e )

Ind o -1 ( V i o l e t )

4 0 5 / 2 0

5 3 0 / 3 0

4 5 0 L P

I n d o - 1 ( B l u e )


DsRed

To use DsRed, replace the filter for the C PMT of the blue octagon as shownbelow.

488-nm blue laser


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4 8 8 /

1 0

5 3 0 / 3 0 5 0 5 L P

F I T C

S S C

6 9 5 / 4 0

6 8 5 LP

7 8 0 / 6 0

7 3 5 L P

5 8 5 / 4 2

5 5 0 L P

P E - C y 7 D s R e d

P e r C P -C y 5.5

PerCP or BD Cy-Chrome Reagent

To use PerCP or BD Cy-Chrome reagent, replace the mirror and filter for the BPMT of the blue octagon as shown below.

488-nm blue laser


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4 8 8 /

1 0

5 3 0 / 3 0 5 0 5 L P

F I T C

S S C

6 7 0 / 1 4

6 3 5 LP

7 8 0 / 6 0

7 3 5 L P

5 7 5 / 2 6

5 5 0 L P

P E - C y 7 P E

P e r C P

Appendix E

Special Order Configurations


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133

The BD LSR II cytometer can be ordered with, or upgraded to, one of severallaser and detector array options. You must use the Cytometer Setup and Trackingapplication to set up new configurations. See the Cytometer Setup and Tracking Applications Guide for more information.

This appendix contains the following information:

Common Special Order Configurations on page 134

Special Order Configuration Trigon and Octagon Maps on page 148

Common Special Order Configurations

The following are commonly used configurations.

6-Blue 0-Violet 0-UV 3-Red Configuration on page 135





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6-Blue 0-Violet 0-UV 3-Red Configuration

6-Blue 0-Violet 0-UV 3-Red supports a blue octagon and a red trigon.Table E-1 shows the detectors, filters, and mirrors used in the defaultconfiguration. The word blank indicates that a blank optical holder shouldbe used instead of a mirror or filter. A dash () indicates that no slot exists fora mirror in that PMT position.

The 6-Blue 0-Violet 0-UV 3-Red maps are:

6-Color Blue Octagon Default Configuration Map on page 149


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Appendix E: Special Order Configurations 135

3-Color Red Trigon Default Configuration Map on page 153

Table E-1 6-Blue 0-Violet 0-UV 3-Red default mirror and filter configuration


PMT(Detector)

Longpass

DichroicMirror

BandpassFilter



A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5

D 600 LP 610/20 PE-Texas RedE 550 LP 575/26 PE

F 505 LP 530/30 FITC, Alexa Fluor 488

G blank 488/10 SSC

H blank none


A 755 LP 780/60 APC-Cy7

B 710 LP 730/45 Alexa Fluor 700

C 660/20 APC


6-Blue 2-Violet 0-UV 3-Red supports a blue octagon, and violet and red trigons.Table E-2 shows the detectors, filters, and mirrors used in the defaultconfiguration. The word blank indicates that a blank optical holder should beused instead of a mirror or filter. A dash () indicates that no slot exists for amirror in that PMT position.




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2-Color Violet Trigon Default Configuration Map on page 150




PMT(Detector)


BandpassFilter



A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5

D 600 LP 610/20 PE-Texas Red

E 550 LP 575/26 PE


G blank 488/10 SSC

H blank none




C blank none


A 755 LP 780/60 APC-Cy7

B 710 LP 730/45 Al Fl 700

Table E-2 6-Blue 2-Violet 0-UV 3-Red default mirror and filter configuration (continued)


PMT(Detector)


BandpassFilter



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6-Blue 0-Violet 2-UV 3-Red supports a blue octagon, and UV and red trigons.Table E-3 on page 138 shows the detectors, filters, and mirrors used in thedefault configuration. The word blank indicates that a blank optical holdershould be used instead of a mirror or filter. A dash () indicates that no slotexists for a mirror in that PMT position.



2-Color UV Trigon Default Configuration Map on page 152



C 660/20 APC



PMT(Detector)

Longpass

DichroicMirror

BandpassFilter



A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5

D 600 LP 610/20 PE T R d


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D 600 LP 610/20 PE-Texas RedE 550 LP 575/26 PE


G blank 488/10 SSC

H blank none


A 505 LP 530/30 Indo-1 (Blue)


C blank none


A 755 LP 780/60 APC-Cy7


C 660/20 APC


6-Blue 2-Violet 2-UV 3-Red supports a blue octagon, and violet, UV, and redtrigons. Table E-4 shows the detectors, filters, and mirrors used in the defaultconfiguration. The word blank indicates that a blank optical holder should beused instead of a mirror or filter. A dash () indicates that no slot exists for amirror in that PMT position.



2 Color Violet Trigon Default Configuration Map on page 150


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PMT(Detector)


BandpassFilter


blue octagon

(488-nm laser)

A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5


E 550 LP 575/26 PE


G blank 488/10 SSC

H blank none




C blank none


A 505 LP 530/30 Indo-1 (Blue)

B blank 450/50 Indo-1 (Violet) DAPI



PMT(Detector)


BandpassFilter



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6-Blue 6-Violet 0-UV 3-Red supports blue violet octagons, and a red trigon.Table E-5 on page 141 shows the detectors, filters, and mirrors used in thedefault configuration. The word blank indicates that a blank optical holder

should be used instead of a mirror or filter. A dash () indicates that no slotexists for a mirror in that PMT position.



6-Color Violet Octagon Default Configuration Map on page 151


B blank 450/50 Indo 1 (Violet), DAPI

C blank none


A 755 LP 780/60 APC-Cy7


C 660/20 APC



PMT(Detector)


BandpassFilter



A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5


E 550 LP 575/26 PE


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E 550 LP 575/26 PE


G blank 488/10 SSC

H blank none

violet octagon

(405-nm laser)

A 630 LP 655/8 Qdot 655

B 595 LP 605/12 Qdot 605

C 575 LP 585/15 Qdot 585

D 545 LP 560/20 Qdot 565

E 475 LP 525/50 AmCyan, Qdot 525

F blank 450/50 Pacific Blue

G blank blank none

H blank none


A 755 LP 780/60 APC-Cy7


C 660/20 APC


6-Blue 6-Violet 0-UV 4-Red supports blue, violet, and red octagons.Table E-6 shows the detectors, filters, and mirrors used in the defaultconfiguration. The word blank indicates that a blank optical holder shouldbe used instead of a mirror or filter. A dash () indicates that no slot exists fora mirror in that PMT position.





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g g p p g

4-Color Red Octagon Default Configuration Map on page 154



PMT(Detector)


BandpassFilter



A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5D 600 LP 610/20 PE-Texas Red

E 550 LP 575/26 PE


G blank 488/10 SSC

H blank none

violet octagon(405-nm laser)

A 630 LP 655/8 Qdot 655

B 595 LP 605/12 Qdot 605

C 575 LP 585/15 Qdot 585

D 545 LP 560/20 Qdot 565




PMT(Detector)


BandpassFilter



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G blank blank none

H blank none

red octagon

(633-nm laser)

A 755 LP 780/60 APC-Cy7


C 675 LP 685/35 Alexa Fluor 680

D 660/20 APC

E blank blank none

F blank blank none

G blank blank none

H blank none


6-Blue 6-Violet 2-UV 3-Red supports blue and violet octagons, and UV and redtrigons. Table E-7 shows the detectors, filters, and mirrors used in the defaultconfiguration. The word blank indicates that a blank optical holder should beused instead of a mirror or filter. A dash () indicates that no slot exists for amirror in that PMT position.





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PMT(Detector)


BandpassFilter


blue octagon

(488-nm laser)

A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5


E 550 LP 575/26 PE


G blank 488/10 SSC

H blank none


A 630 LP 655/8 Qdot 655

B 595 LP 605/12 Qdot 605

C 575 LP 585/15 Qdot 585

D 545 LP 560/20 Qdot 565



G blank blank none

H blank none



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H blank none


A 505 LP 530/30 Indo-1 (Blue)


C blank none

red trigon(633-nm laser) A 755 LP 780/60 APC-Cy7B 710 LP 730/45 Alexa Fluor 700

C 660/20 APC


6-Blue 6-Violet 2-UV 4-Red supports blue, violet, and red octagons, and a UVtrigon. Table E-8 shows the detectors, filters, and mirrors used in the defaultconfiguration. The word blank indicates that a blank optical holder should beused instead of a mirror or filter. A dash () indicates that no slot exists for amirror in that PMT position.





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PMT(Detector)


BandpassFilter


blue octagon

(488-nm laser)

A 755 LP 780/60 PE-Cy7

B 685 LP 695/40 PerCP-Cy5.5

C 655 LP 660/20 PE-Cy5


E 550 LP 575/26 PE


G blank 488/10 SSC

H blank none


A 630 LP 655/8 Qdot 655

B 595 LP 605/12 Qdot 605

C 575 LP 585/15 Qdot 585

D 545 LP 560/20 Qdot 565

E 475 LP 525/50 AmCyan, Qdot 525F bl k 450/50 P ifi Bl



PMT(Detector)


BandpassFilter



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G blank blank none

H blank none

UV trigon

(355-nm laser)

A 505 LP 530/30 Indo-1 (Blue)


C blank none

red octagon(633-nm laser)

A 755 LP 780/60 APC-Cy7


C 675 LP 685/35 Alexa Fluor 680

D 660/20 APC

E blank blank none

F blank blank none

G blank blank none

H blank none

Special Order Configuration Trigon andOctagon Maps

If a slot contains a filter or mirror, a number appears in the correspondingposition on the configuration map. If a slot contains a blank optical holder,nothing is written in the corresponding position on the configuration map.

The default configuration maps are:




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6-Color Blue Octagon Default Configuration Map

6 1 0 / 2 0

P E - T e x a s R e d

6 9 5 / 4 0

6 8 5 LP

P e r C P -C y 5.5

488-nm blue laser


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5 7 5 /

2 6

0 6 0 0 L P

P E

5 3 0 / 3 0

5 0 5 L

P

5 5 0

L P

4 8 8 / 1 0

7 8 0 / 6 0

7 5 5 L P

6 6 0 / 2 0

6 5 5 L P

P E - C y 7 P E - C y

5

S S C

F I T C

2-Color Violet Trigon Default Configuration Map

4 5 0 / 5 0

P ac i f i c Bl ue

405-nm violet laser


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5 2 5 / 5 0

5 0 5 L P

A m C y a n

6-Color Violet Octagon Default Configuration Map

5 6 0 / 2 0

Q d o t 5 6 5

6 0 5 / 1 2

5 9 5 LP

Q d o t 60 5

405-nm violet laser


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5 2 5 /

5 0

5 4 5 L P

A m C y a n

4 5 0 / 5 0

4 7 5

L P

P a c i f i c B l u e

6 5 5 / 8

6 3 0 L P

5 8 5 / 1 5

5 7 5 L P

Q d o t 6 5 5 Q d o t 5

8 5

2-Color UV Trigon Default Configuration Map

4 5 0 / 5 0


355-nm UV laser


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5 3 0 / 3 0

5 0 5 L P

I n d o - 1 ( B l u e )

3-Color Red Trigon Default Configuration Map

7 3 0 / 4 5

7 10 LP

Al e x a F l uo r 7 0 0

633-nm red laser


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7 8 0 / 6 0

7 5 5 L P

6 6 0 / 2 0

A P C - C y 7 A P C

4-Color Red Octagon Default Configuration Map

6 6 0 / 2 0

A P C

7 3 0 / 4 5

7 10 LP

Al e x a F l uo r 7 0 0

633-nm red laser


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7 8 0 / 6 0

7 5 5 L P

6 8 5 / 3 5

6 7 5 L P

A P C - C y 7 A l e x a F l u

o r 6 8 0

Appendix FSetting Laser Delay

This appendix describes how to optimize laser delay settings in a multiple laser


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155

system.

About Laser Delay on page 156

Optimizing Laser Delay on page 157

About Laser Delay

Sample interrogation takes place within the cuvette flow cell. Laser light isdirected through a series of prisms that focus multiple lasers on the event stream

at different positions. This allows optimal detection of fluorescent signal fromeach laser with minimal cross-contamination from the other beams.

In the BD LSR II four-laser system, the blue laser intercepts the stream first,followed by the violet, UV, and red lasers. Because the laser signals are spatiallyseparated, there is a slight delay between the detection of each lasers signal(Figure F-1 ).

Figure F-1 Signal separation over time

event intercepts red laser


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The laser delay setting in BD FACSDiva software is used to realign the signals sothey can be measured and displayed on the same time scale. Signals are alignedwith respect to the blue laser, so the blue laser will have a 0 delay value, and thered laser will have the longest delay.

time

red

blue

UV

violet

event intercepts blue laser

event intercepts violet laser

event intercepts UV laser

event intercepts red laser

Optimizing Laser Delay

Laser delay is set using BD FACSDiva software. To optimize the delay for a givenlaser, you acquire events from a sample with a fluorescence signal excited by that

laser. Follow the procedures in Running Samples on page 53 , for sampleoptimization and acquiring data.

To optimize laser delay:

1 While acquiring data from your sample, create a histogram to show thefluorescence signal excited by the laser in which the delay is to beoptimized.

2 In the Acquisition Dashboard, set the Events to Display to 500 evt.


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Appendix F: Setting Laser Delay 157

3 Select the Laser tab in the Cytometer window.

Window extension and laser delay values are displayed in microseconds(sec).

Figure F-2 Laser tab of the Cytometer window

4 Set the window extension value to 0 sec.

5 Set an initial laser delay value ONLY for the laser you are optimizing.

If you are optimizing the violet laser, set its delay to 20 sec.

If you are optimizing the UV laser, set its delay to 40 sec.

If you are optimizing the red laser, set its delay to 60 sec.

6 While observing the positive events on the histogram, adjust the laser delayin 1 sec increments within a range of 10 sec of the initial setting.

Choose the setting that moves the events farthest to the right (highestfluorescence intensity).

7 Draw an interval gate on the histogram for the positive events.

8 Create a statistics view to display the mean fluorescence intensity of thegated population.


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g p p

9 While observing the mean fluorescence intensity for the gated population,adjust the laser delay in 0.1 sec increments within a range of 2.0 sec of the setting obtained in step 6 .

You should also stay within a range of 10 sec of the initial setting(see step 5 ).

Preserve the setting that maximizes the fluorescence intensity.

10 Reset the window extension to 10 sec.

IndexAacridine orange (AO) 81adjusting

threshold 63voltages 63air in filter, removing 50l i 31

BD LSR IIcomponents 26cytometer optics 55online Help xiiworkstation 38

beam splitters See dichroic mirrors.biohazards xx, 31


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159

alarm, waste container 31ALPHA filters (AFs) 35analysis

data 68immunophenotyping 72reusing 77saving 77

antibodies 106arm, tube support 30assistance, technical xv

BBal seal, replacing 90bandpass (BP) filters 35, 109

holder 42specifications 45

battery test 85Battery Test switch, shown 85battery, changing 86BD FACSClean solution 81BD FACSDiva software See softwareBD FACSFlow solution 47BD FACSRinse solution 81BD Falcon tubes 122

blank optical holders 42bleach solution 81blue laser 34bubbles, removing air 50

buttonsfluid control 28sample flow rate control 28

Ccalculating compensation 66, 67capacity, waste container 31changing battery 86Class I laser product xviiiCoherent lasers 34compensation

calculating 66, 67theory 111tubes, creating 61

componentscytometer shown 26optical bench 32

computer system, about 38configuration maps 126

containers, sheath and waste 31, 46, 49control panel, cytometer 27controls

compensation 61single-stained 55, 68

cord, damaged xixcovers, cytometer 26creating

analysis objects 72compensation tubes 61global worksheets 69statistics view 73

custom configurations 130cuvette flow cell 158CV, troubleshooting 100Cy-Chrome, filter configuration for 134

detectorsphotodiode 37photomultiplier tubes (PMTs) 37sensitivity 37

dichroicfilters 35mirrors 35, 111

digital data 26discriminating filters (DFs) 110DNA

flow rate for analysis 104droplet containment system 30

troubleshooting 94DsRed, filter configuration for 133

E
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cytometer 26components shown 26configuration 43, 55covers and door 26, 44

dimensions xiiihandles 27, 32setup 54spares kit 130symbols and labels xxi

D

DAPIcleaning after using 81

dataanalyzing 68, 72gating 72recording 68, 70

debris

excessive 100removing 63delay, laser 158

Eelectrical

requirements xiiisafety xix

electronics 114emission duration 114environmental requirements xiiievent rate

erratic 98, 99high 97low 98zero 95, 96, 97

excessive debris 100excitation wavelength 106experiments

immunophenotyping 69sample optimization 58

extra filters and mirrors 130

FFalcon tubes 94

filtersbandpass 35optical

bandpass 35, 109changing 44dichroic 35discriminating 110extra 130longpass 35, 108shortpass 35, 109specifications 45theory 107

sheathreplacing 88

FITC and Stokes shift 106fixed-alignment lasers 26

Ggating data 72global worksheets 72

creating 69previewing data 68, 77

Hhandles, cytometer 27, 32hazards

biological xx, 31electrical xix

laser xviiHoechst 81hydrodynamic focusing 104
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161

flow cell 104draining 51

flow rate control buttons 28fluid control buttons 28

fluidics 104cleaning 80description 28, 104flow rate control 28flushing system 83priming 51removing air bubbles 50run mode 28sample injection port (SIP) 29, 30

fluidics interconnect 50fluorescence 106

emission 114fluorochromes 111

emission spectra 107filter configurations and 124, 130

role in light emission 106which measured 43

forward scatter (FSC) 33, 34, 105FSC and SSC voltages, adjusting 63FSC threshold, adjusting 63

Iimmunophenotyping 104

analysis 72

data 69experiment 69

indo-1, filter configuration for 132

J JDS Uniphase HeNe 34

Kknob

SAMPLE FINE ADJ 28sheath clamp, shown 46

Llabels on cytometer xxilaser delay 157

lasersabout 34classification xviiipower 34quality control (QC) 52, 120safety xviiwarming up 40warmup times 34

longpass (LP) filters 35, 108longpass dichroic mirrors

holder 42specifications 45

LSR II See cytometer, BD LSR II.

Mmaintenance

Ooctagon

description 32detector 26location 44shown 36

optical bench components 32optics 55

components 32cover 33custom filter configurations

DsRed 133

indo-1 132PerCP or BD Cy-Chrome 134PE-Texas Red 131

dichroic mirrors 35 111
http://-/?-http://-/?-http://-/?-http://-/?-


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maintenancefluidics

daily cleaning 80flushing system 83

periodicBal seal 90replacing sheath filter 88sample tube O-ring 92

scheduledbattery change 86battery test 85system flush 83waste management system 85

Microsoft Windows operatingsystem 38

mirrorsdichroic longpass 35extra 130specifications 45

NNCCLS documents xx

dichroic mirrors 35, 111filters 35, 44, 107location 26steering 32, 36

optimizationsample 54

ordering spare parts 119O-ring, sample tube, replacing 92

PPerCP

filter configuration for 134Stokes shift 106PE-Texas Red 131photodiode 37photomultiplier tubes (PMTs) 32, 37population hierarchy 72power

requirements xixswitch 26

priming fluidics system 51propidium iodide (PI)

cleaning after using 81

pulsedefinition 114measurements 116processors 116

Qquality control (QC)

about 52particles 120troubleshooting 101

RRadius laser 34recording

compensation Tubes 65, 66

sampleinjection tube 30optimization 54

experiment 58single-stained controls 55, 68

tube requirements 94SAMPLE FINE ADJ knob 28sample injection port (SIP) 104

cleaning 80components 29, 30problems with 94replacing Bal seal 90replacing sample tube O-ring 92

samples, running 70Sapphire laser 34saving analyses 77scatter
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163

co pe sat o ubes 65, 66data 68, 70

red laser 34removing air bubbles, filter 50replacing

Bal seal 90battery 86optical filters 44sample tube O-ring 92sheath filter 88

requirements specifications xiiireusing analyses 77

Ssafety

biological xxelectrical xixgeneral xxilaser xviisymbols and labels xxi

scatterlight 105parameter distortion 99

setting laser delay 157

setup, cytometer 54sheathcontainer 31, 46filter

replacing 88flow 104fluid 104

backflush 30pressure 104

sheath clamp knob 46sheath container

cap 47closing 47depressurize 47

shortpass (SP) filters 35, 109

Show Population Hierarchy 73shutdown, cytometer 80side scatter (SSC) 34, 105signals, amplifying 37SIP See sample injection port.

softwareadjusting detector voltages 37cytometer control xi

sparesfilters and mirrors 130kit 130parts, ordering 119

specifications, filter and mirror 45spillover 112statistics views 72steering optics 32, 36Stokes shift 106switch, Battery Test 85

symbols on cytometer xxi

T

Vviolet laser 34virus protection software xxiiivoltages, PMT, adjusting 63

Wwaste container 31, 46

alarm 31capacity 31emptying 48

window extension 159workstation, about 38
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technical assistance xvTechnical Assistance, assistance

technical xvtemperature requirements xiiitesting battery 85text conventions xithiazole orange (TO) 81threshold

adjusting 63defined 117

trigon 37description 32detector 26location 44

tubescompensation 61requirements 94

Uungrounded receptacles xixuser preferences 56UV laser 34

Bd Lsrii User's Guide

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