Steps to Success with Multicolor Flow Cytometry Holden T. Maecker.

Steps to Success with

Multicolor Flow Cytometry

Holden T. Maecker

Holden Maecker, Flow Cytometry Consulting

Outline

1. Configure your instrument

2. Characterize your instrument

3. Design your panel

4. Optimize settings for your panel

5. Run appropriate controls

6. QC your data


Outline

1. Configure your instrument Number and type of lasers Number of PMTs per laser Choice of filters and dichroic mirrors

These choices will determine: What fluorochromes you can use effectively How well certain fluorochrome combinations

will perform


How do we measure performance?

W2

W1

D

Where D = difference between positive and negative peak medians, andW = 2 x rSD (robust standard deviation)

Stain Index = D / W

Resolution Sensitivity:


CD127 PE

300 400 500 600 7000

5

10

15

20

25

30

35

40

25 mW green laser (532 nm)

100 mW blue laser (488 nm)

25 mW blue laser (488 nm)

PMT voltage

Sta

in i

nd

ex

An Example: Green vs. Blue Lasers

Green laser more efficient for PE and PE tandems Green laser less efficient for FITC, PerCP and GFP


Second Example: Filters and Spillover


Outline

2. Characterize your instrument Obtain minimum baseline PMT settings Track performance over time

This will allow you to: Run the instrument where it is most sensitive Be alert to changes in the instrument that

might affect performance


SDEN

Baseline PMTV is set by placing the dim bead MFI to equal 10X SDBaseline PMTV is set by placing the dim bead MFI to equal 10X SDENEN

460 V

Automated baseline PMT voltage determination in Diva 6.0


Performance Tracking

A variety of parameters can be tracked: Linearity, CVs, laser alignment PMT voltages required to hit target values

Data can be visualized in Levey-Jennings plots:

400

425

450

475

500

525

550

10/22/04 11/11/04 12/01/04 12/21/04 01/10/05 01/30/05 02/19/05 03/11/05

Time

PM

T V

olt

ag

e

FITC Channel (Blue laser)FITC Channel (Blue laser)


Outline

3. Design your panel Reserve brightest fluorochromes for

dimmest markers and vice versa Avoid spillover from bright populations into

detectors requiring high sensitivity Beware of tandem dye issues Titrate antibodies for best separation

This will allow you to: Maintain resolution sensitivity where you

most need it Avoid artifacts of tandem dye degradation


Various fluorochromes-stain index

Reagent Clone Filter Stain Index

PE RPA-T4 585/40 356.3

Alexa 647 RPA-T4 660/20 313.1

APC RPA-T4 660/20 279.2

PE-Cy7 RPA-T4 780/60 278.5

PE-Cy5 RPA-T4 695/40 222.1

PerCP-Cy5.5 Leu-3a 695/40 92.7

PE-Alexa 610 RPA-T4 610/20 80.4

Alexa 488 RPA-T4 530/30 75.4

FITC RPA-T4 530/30 68.9

PerCP Leu-3a 695/40 64.4

APC-Cy7 RPA-T4 7801/60 42.2

Alexa 700 RPA-T4 720/45 39.9

Pacific Blue RPA-T4 440/40 22.5

AmCyan RPA-T4 525/50 20.2


Spillover affects resolution sensitivity

Without CD45 AmCyan: With CD45 AmCyan:

CD19 FITC

Note that this is only an issue when the two markers (CD45 and CD19) are co-expressed on the same cell population.


Special requirements of tandem dyes

Compensation requirements for tandem dye conjugates can vary, even between two experiments with the same antibody Degrade with exposure to light, temperature, and

fixation Stained cells are most vulnerable

Solutions: Minimize exposure to above agents Use BD stabilizing fixative if a final fix is necessary Run experiment-specific compensation


False positives due to tandem degradation

A.

False positives inAPC channel reducedin absence of APC-Cy7

False positivesin PE channelremain

Gating scheme CD8 APC-Cy7+ cells CD4 PE-Cy7+ cells

B.

With CD8 APC-Cy7 and CD4 PE-Cy7:

Without CD8 APC-Cy7:


New tandems can be more stable

APC-H7 as a replacement for APC-Cy7:

Comparison of Sample Stability

(in BD Stabilizing Fixative at RT)

0

50

100

150

200

250

0 1 2 4 6 8 24 48

Hours of light exposure

% S

pillo

ver

CD4 APC-H7

CD8 APC-H7

CD4 APC-Cy7

CD8 APC-Cy7


Antibody titration basics

For most purposes, the main objective is to maximize signal:noise (pos/neg separation) This may occur at less than saturated staining This may or may not be the manufacturer’s

recommended titer Titer is affected by:

Staining volume (e.g., 100 L) Number of cells (not critical up to ~5x106) Staining time and temperature (e.g., 30 min RT) Type of sample (whole blood, PBMC, etc.)


Antibody titration example

11010010001

10

100

1000

10000 signal

noise

S:N

ng antibody


Outline

4. Optimize settings for your panel Derive experiment-specific PMT settings Run compensation controls for each

experiment

This will allow you to: Use settings most appropriate for your panel Avoid gross errors of compensation


Experiment-specific setup for a new panel

1. Set voltages to achieve baseline target values

2. Run single-stained CompBeads to see if each bead is at least 2x brighter in its primary detector vs. other detectors• If not, increase voltage in the primary detector (beware: potential

reagent problem)

3. Run fully-stained cells and:• Decrease voltages for any detectors where events are off-scale• Increase voltages for any detectors where low-end resolution is

poor (theoretically should not be necessary)

4. Re-run single-stained CompBeads and calculate compensation

5. Re-run fully-stained cells and repeat step 3 (if further changes made, re-run compensation)

6. Save experiment-specific settings as target values

7. Run samples


Experiment-specific setup for existing panel

Set voltages to achieve experiment-specific target channels

Run single-stained CompBeads and calculate compensation

Run samples


Outline

5. Run appropriate controls Instrument setup controls (e.g., CompBeads) Gating controls (e.g., FMO) Biological controls (e.g., unstimulated

samples, healthy donors)

This will allow you to: Obtain consistent setup and compensation Gate problem markers reproducibly Make appropriate biological comparisons

and conclusions


CompBeads as single-color controls

CompBeads provide a convenient way to create single-color compensation controls:

• Using the same Abs as in the experimental samples

• Creating a (usually) bright and uniform positive fluorescent peak

• Without using additional cells


Frequent compensation questions

Do I need to use the same antibody for compensation as I use in the experiment?

Yes, for certain tandem dyes (e.g., PE-Cy7, APC-Cy7)

Are capture beads better than cells for compensation?

Usually, as long as the antibody binds to the bead and is as bright or brighter than stained cells

Should compensation controls be treated the same as experimental samples (e.g., fixed and permeabilized)?

Yes, although with optimal fix/perm protocols this may make little difference


Comparison of gating controls


Consider using lyophilized reagents

Lyophilization provides increased stability, even at room temperature or 37oC

One batch of reagents can be used for an entire longitudinal study

Pre-configured plates can avoid errors of reagent addition

Complex experiments (multiple stimuli, multiple polychromatic staining cocktails) become easier

Lyophilized cell controls can provide run-to-run standardization


Outline

6. QC your data Visually inspect compensation Visually inspect gating Set sample acceptance criteria

This will allow you to: Avoid classification errors and false

conclusions due to improper compensation and/or gating, or sample artifacts


Visually inspect compensation

Create a template containing dot plots of each color combination in your experiment, then examine a fully stained sample for possible compensation problems

Yikes!


Visually inspect gating

Check gating across all samples in the experiment

Gates may need to be adjusted across donors and/or experimental runs; dynamic (e.g., snap-to) gates may help in some cases

IL-2 PE

IFN

FIT

C


Types of sample acceptance criteria

Minimum viability and recovery for cryopreserved PBMC

Minimum number of events collected in an appropriate gate (e.g., lymphocytes)

Minimum number of events within a region of interest, to calculate an accurate percentage


Outline

1. Configure your instrument

2. Characterize your instrument

3. Design your panel

4. Optimize settings for your panel

5. Run appropriate controls

6. QC your data


A question for you to answer

How many colors can you combine and still have robust results? This depends on:

-The experimental question

-The instrument used

-The markers to be combined


References

Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J. 2004. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62: 169.

Maecker, H. T., and Trotter, J. 2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69: 1037.

Roederer, M. 2008. How many events is enough? Are you positive? Cytometry A 73: 384-385.

McLaughlin, B. E., N. Baumgarth, M. Bigos, M. Roederer, S. C. De Rosa, J. D. Altman, D. F. Nixon, J. Ottinger, C. Oxford, T. G. Evans, and D. M. Asmuth. 2008. Nine-color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach. Cytometry A 73: 400-410.


Acknowledgements

Laurel Nomura Margaret Inokuma Maria Suni Maria Jaimes Smita Ghanekar Jack Dunne Skip Maino

Joe Trotter Dennis Sasaki Marina Gever

Steps to Success with Multicolor Flow Cytometry Holden T. Maecker.

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