Aurora User Training July 2019 Cytometry and Antibody Technology - - PowerPoint PPT Presentation

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Aurora User Training July 2019 Cytometry and Antibody Technology - - PowerPoint PPT Presentation

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Aurora User Training July 2019 Cytometry and Antibody Technology Facility University of Chicago What is spectral cytometry and how is it different than conventional flow cytometry? Cytometry and Antibody Technology Facility Aurora Training

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SLIDE 1

Aurora User Training

July 2019 Cytometry and Antibody Technology Facility University of Chicago

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SLIDE 2

What is spectral cytometry and how is it different than conventional flow cytometry?

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 3

First, let’s review conventional cytometry

  • In conventional cytometry, one detector is assigned to one

fluorophore

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 4

Is a fluorochrome only the section of spectra that we choose to view?

Wavelength Emission Intensity APC Emission Spectrum 670/30 Filter

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 5

With spectral cytometery, all detectors are used for all fluorophores

–

Laser Channel Center Wavelenght (nm) Bandwith (nm) Start (nm) End (nm) B1 508 20 498 518 B2 524.5 17 516 533 B3 541.5 17 533 550 B4 580.5 19 571 590 B5 598 20 588 608 B6 615 20 605 625 B7 660 17 652 669 B8 678 18 669 687 B9 697 19 688 707 B10 717 20 707 727 B11 738 21 728 749 B12 760 23 749 772 B13 783 23 772 795 B14 811.5 34 795 829 Blue Laser Laser Channel Center Wavelenght (nm) Bandwith (nm) Start (nm) End (nm) V1 427.5 15 420 435 V2 443 15 436 451 V3 458 15 451 466 V4 473 15 466 481 V5 508 20 498 518 V6 524.5 17 516 533 V7 541.5 17 533 550 V8 580.5 19 571 590 V9 598 20 588 608 V10 615 20 605 625 V11 664 27 651 678 V12 691.5 28 678 706 V13 720 29 706 735 V14 749.5 30 735 765 V15 779.5 30 765 795 V16 811.5 34 795 829 Violet Laser Channel Center Wavelenght (nm) Bandwith (nm) Start (nm) End (nm) R1 660 17 652 669 R2 678 18 669 687 R3 697 19 688 707 R4 717 20 707 727 R5 738 21 728 749 R6 760 23 749 772 R7 783 23 772 795 R8 811.5 34 795 829 Red Laser Laser Channel Center Wavelenght (nm) Bandwith (nm) Start (nm) End (nm) YG1 577 20 567 587 YG2 598 20 588 608 YG3 615 20 605 625 YG4 660 17 652 669 YG5 678 18 669 687 YG6 697 19 688 707 YG7 720 29 706 735 YG8 749.5 30 735 765 YG9 779.5 30 765 795 YG10 811.5 34 795 829 Yellow Green

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 6

With spectral cytometry, each fluorophore is identified by their distinct signature

The entire emission spectra of fluorescent dyes excited by the

  • nboard lasers is measured

Emission spectra excited by the Violet, Blue, and Red lasers are measured from the laser line to the infrared region. Full spectrum capture enables the use of novel unmixing algorithm for data analysis. BV785 APC PE Violet Blue Red Yellow Green

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 7

Spectral cytometry allows us to use more colors because we can separate highly overlapping fluorophores

APC Alexa Fluor 647

Cytometry and Antibody Technology Facility Aurora Training Course

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What is the workflow?

  • Conventional flow cytometry
  • 1. Run single stained controls
  • 2. Set voltages
  • 3. Compensate (optional at this

step)

  • 4. Run full stained samples
  • 5. Analyze samples in FlowJo or

FCS Express

  • Compensation can be done after

sample collection

  • Spectral flow cytometry
  • 1. Run unstained cells
  • 2. Run reference controls (single

stains)

  • 3. Spectral unmixing

(recommended but optional at this step)

  • 4. Run full stained samples
  • 5. Analyze samples in FlowJo or

FCS Express

  • 6. (Spectral unmixing can be

done again if needed)

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 9

The unmixing algorithm converts raw data to unmixed data

Unmixi xing Al Algorithm

Raw Worksheet Unmixed Worksheet

Cytometry and Antibody Technology Facility Aurora Training Course

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Raw vs. Unmixed Data

Raw Data

  • Parameters are the

instrument channels (V1, V2, etc.)

  • Visualized in raw worksheet
  • Large fcs file size: at least 48

parameters + FSC and SSC

  • Can be unmixed as many

times as desired Unmixed Data

  • Parameters are the

fluorochromes included in the assay

  • Visualized in unmixed

worksheet

  • Smaller fcs file size: number
  • f fluors + FSC and SSC
  • Can not be used to unmix

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 11

How does the unmixing work?

0.05 0.1 0.15 0.2 0.25 0.3 0.35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fluorophore 1

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fluorophore 2

100 200 300 400 500 600 700 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fluorophore 1 & 2

How do we determine how much of each fluorophore is contributing to this signal?

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 12

How does the unmixing work?

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 13

How does the unmixing work?

0.19 0.21 0.32 0.001 0.2 0.03 0.12 0.39 0.06 0.28 0.03 0.12 0.01 0.08 0.005 0.03 0.01 0.005

Mixing Matrix

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 14

How does the unmixing work?

0.19 0.21 0.32 0.001 0.2 0.03 0.12 0.39 0.06 0.28 0.03 0.12 0.01 0.08 0.005 0.03 0.01 0.005

Mixing Matrix (M)

39.83 420.89 643.28 446.61 601.32 387.94 175.17 109.03 44.05 9.71 5.36 0.83

Abundances (!) Observed (")

#$ = &

Solve for $. [Fluorophore 1 and Fluorophore 2] (Unknowns)

Cytometry and Antibody Technology Facility Aurora Training Course

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Reference Controls

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 16

Reference Controls

  • In order to get successful unmixing, you need to have excellent

reference controls

  • Garbage in = garbage out!!

Cytometry and Antibody Technology Facility Aurora Training Course

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Good Controls = Good Data

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Optimal controls are needed for accurate unmixing/compensation: Positive and negative particles clearly separated Negative and positive particles with IDENTICAL autofluorescence characteristics Sufficient events for both data points Fluorescence spectrum of positive control needs to be IDENTICAL to the one in the multicolor sample

Fluor A Fluor B

X X (-) (+)

Slides from Cytek

A positive signal from cells cannot be matched with a negative signal from beads. A minimum of 200 events for the positive and negative populations is required. More is better.

v The positive signal intensity of the control must be as bright or brighter than the multicolor sample

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 18

Fluorescence spectrum of positive control needs to be IDENTICAL to the one in the multicolor sample

  • For any tandem dye, you must use the exact same antibody – even

the same lot

  • For a non tandem dye, you could potentially use a different antibody

with the same fluorophore

  • You can’t use “equivalent” fluorophores for your controls
  • FITC is not the same as GFP
  • BV510 is not the same as live/dead aqua dye
  • Special considerations for lineage/dump channels if using tandem

dyes

  • Fix/perm buffers can alter the spectra of a fluorophore, so all controls

must be treated with the same buffers as your sample

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 19

Reference Control Multicolor Sample Will it unmix correctly? PD-L1 PE-Dazzle 594 on cells PD-L1 PE-Dazzle 594 Yes PD-L1 PE-Dazzle 594 on compensation beads PD-L1 PE-Dazzle 594 Yes CD4 BV750 FoxP3 BV750 No BV421, BV480 and BV510 are the only non-tandem BV Fluorophores CD4 PE Tetramer PE Yes CD69 APC on unstimulated cells CD69 APC on activated cells No RC is probably dimmer than multicolor sample

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 20

Spectrum Plots Allow to QC Controls (1)

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Expected Spectrum Clean Background

Slides from Cytek If you are using compensation beads, please wash them after staining

Cytometry and Antibody Technology Facility Aurora Training Course

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Spectrum Plots Allow to QC Controls

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When gated in the peak of the distribution, spectrum looks normal When gated on the brightest portion

  • f the distribution, spectrum looks like

Super Bright 436

Spectrum Plots Allow to QC Controls

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When gated in the peak of the distribution, spectrum looks normal When gated on the brightest portion

  • f the distribution, spectrum looks like

Super Bright 436

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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What can we see in the spectra?

  • If your antibodies are too old and degrading – Ex: Cy7 is falling
  • ff of APC
  • If you accidentally contaminated your reference control or

antibody stock with another fluorophore

  • If you accidentally mislabeled your reference control and it is

actually for a different fluorophore

  • Of if you have two different reference controls that are actually stained

with the same fluorophore

If any of these things happen, the spectral unmixing will be incorrect and your data will look odd!

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 23

Tips for reference controls

  • You may need to “manufacture” reference controls for cells
  • Stimulate cells
  • Use a different tissue/cell line with better marker expression
  • Reccomended live/dead stain control
  • Kill some cells, stain with Zombie NIR, wash, right before running the

control spike in some unstained cells

  • Many ways to kill cells – I like 65°C for 10 minutes

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 24

Should you use compensation beads or cells?

CD62L APC on cells CD62L APC on beads Compensation beads can give a cleaner signature than cells for some markers. BUT beads are not cells, and sometimes beads do not provide accurate unmixing.

Cytometry and Antibody Technology Facility Aurora Training Course

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Beads vs Cells as Controls

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Beads are easy to use. HOWEVER in order for them to be optimal controls:

  • Fluorochrome spectrum needs to be IDENTICAL when antibody is bound to cells vs beads
  • Beads need to be just as bright or brighter than cells
  • 0.20

0.40 0.60 0.80 1.00 1.20 V1 V3 V5 V7 V9 V11 V13 V15 B1 B3 B5 B7 B9 B11 B13 R1 R3 R5 R7 Beads AF488 Cells AF488

  • 0.20

0.40 0.60 0.80 1.00 1.20 V1 V3 V5 V7 V9 V11V13V15 B1 B3 B5 B7 B9 B11B13 R1 R3 R5 R7 Beads BV711 Cells BV711

  • 0.20

0.40 0.60 0.80 1.00 1.20 V1 V3 V5 V7 V9 V11V13V15 B1 B3 B5 B7 B9 B11B13 R1 R3 R5 R7 Beads PerCPeF710 Cells PerCPeF710

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 26

Spectrum Mismatch = Unmixing Inaccuracy

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  • 0.20

0.40 0.60 0.80 1.00 1.20 V1 V3 V5 V7 V9 V11 V13 V15 B1 B3 B5 B7 B9 B11 B13 R1 R3 R5 R7 Beads PerCPeF710 Cells PerCPeF710

Unmixed beads Unmixed cells Use BV711 beads as control Use PerCP-eFluor 710 beads as control

  • 0.20

0.40 0.60 0.80 1.00 1.20 V1 V3 V5 V7 V9 V11 V13 V15 B1 B3 B5 B7 B9 B11 B13 R1 R3 R5 R7 Beads BV711 Cells BV711

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 27

Should I use compensation beads?

  • Yes and no.
  • For best results, stain a full set of cells and compensation

beads for your reference controls

  • Ex: for a 10 marker panel + live/dead, you need 10 tubes for comp

beads and 11 tubes for cells

  • After running the experiment, play around with different combinations of

unmixing: all beads, all cells, mixture of both (usually the best)

  • Determine what works best to unmix that specific panel and use those

controls for subsequent experiments

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 28

Does it matter which compensation beads I use?

  • I tested two of the ThermoFisher compensation beads
  • UltraComp eBeads: positive and negative beads in one vial
  • AbC total antibody: separate vials for positive and negative beads
  • (Also tested the Arc-reactive beads for fixable live/dead dyes but

negative beads are a problem??? Still testing them out)

  • Experiment setup:
  • Mouse spleen cells, UltraComp beads, and AbC total beads were

stained with 0.1 µg of CD4 (clone GK1.5)

  • For the “full stain” tube, I took some cells from all of the single stains

and combined them into one tube. So I expect there to only be single positive cells (since all of the antibodies are CD4)

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 29

Unmixing a “fully stained” sample with different reference controls (mixed multiple CD4 single stains)

”Full stain” cells were unmixed with: Cells AbC Total UltraComp

Conclusion: UltraComp beads seem to win over AbC total beads

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 30

Panel Design

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 31

THE KEY FOR SUCCESFUL PANEL DESIGN

GATHER AS MUCH INFORMATION AS POSSIBLE AROUND THE BIOLOGY OF THE ASSAY! Two main pieces of information:

  • Antigen classification (primary, secondary, tertiary)
  • Antigen co-expression

4

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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Antigen Classification

  • Primary: on or off (examples: CD3, CD4, CD45)
  • Secondary: continuum (examples: CD45RA,

HLA-DR)

  • Tertiary: low or unknown expression (examples:

FoxP3, ICOS)

  • If you are unsure about expression, you can check the data

provided on vendor websites for your antibody of interest (BioLegend, BD, eBioscience/Thermofisher, Miltenyi)

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 33

Antigen co-expression

  • What markers are you using to define/identify populations?
  • What markers characterize these populations once you have

identified them (such as activation status)?

  • What markers are on a lot of cell types? (example: CD45, CD3

in whole blood)

  • What markers are on a small number of cell types? (example:

CD56, CD25 in whole blood)

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 34

Examples on How to Summarize and Provide this Information

5

What about a schematic?

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 35

Examples on How to Summarize and Provide this Information

6

What about a simpler schematic?

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 36

Maybe You Already Have Some Information

7

Data from literature, or a similar assay run in your laboratory/institution? Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 37

Maybe You Have Lots of Valuable Information

8

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 38

Other considerations regarding fluorophores

  • What fluorochromes can I use for this panel?
  • How many antigens do I want to detect?
  • What fluorophores are best for that number of antigens?
  • Avoid using several fluorophores with excessive spill for smaller panels, save

those colors for when you don’t have other options. (Example: don’t use both BV421 and eFluor450 in an 8 color panel)

  • What antibodies are commercially available for each antigen?
  • What fluorophores are available?
  • Are there multiple clones available? Is one clone better than the
  • thers?

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 39

How to assign fluorophores?

  • If possible, you can start with Cytek’s panel and swap out markers that you aren’t using

Specificity Fluorophore Specificity Fluorophore Specificity Fluorophore 3.5 uL CD11c BB515 (BD#564490) 3.5 uL CD27 APC (BL#356410) 3.5 uL CCR7 BV421 (BL#353208) 3.5 uL CD45RA Alexa Fluor 488 (BL#304114) 3.5 uL CD123 Alexa Fluor 647 (BL#306024) 3.5 uL CD19 Super Bright 436 (TF#62-0199-42) 3.5 uL CD3 Alexa Fluor 532 (TF#58- 0038-42) 3.5 uL CD127 APCR 700 (BD#565185) 3.5 uL CD16 eFluor450 (TF#48- 0168-42) 3.5 uL CD25 PE (BL#356104) 3.5 uL HLA DR APC-Fire 750 (BL#307658) 2 uL TCR gamma delta BV480 (BD#566076) 3.5 uL IgD PE/Dazzle 594 (BL#348240) Zombie NIR (titer before using!) BL#423105 3.5 uL CD14 BV510 (BL#301842) 3.5 uL CD95 PE-Cy5 (BL#305610) 3.5 uL CD8 BV570 (BL#301038) 3.5 uL CD11b PerCPCy55 (BL#301328) 3.5 uL CD1c BV605 (BL#331538) 3.5 uL CD38 PerCP-eFluor710 (TF#46-0388-42) 3.5 uL PD-1 BV650 (BL#329950) 3.5 uL CD57 PE-Cy7 (BL#359624) 3.5 uL CD56 BV711 (BL#318336) 2 uL CD4 BV750 (BD#566355) 3.5 uL CD28 BV785 (BL#302950)

Panel from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 40

How to assign fluorophores?

  • Use the tools specifically made for the aurora!!!

To read this table: spread of fluor in the row impacts resolution of the fluor in the column. Red means the fluor in that row has significant spread into the dye in the column (for example PE into AF532). Areas in bright pink and red indicate pairs for which more attention to panel design is needed.

Spread matrix for 24 fluors that can be use in combination

BV421 SB436 eF450 BV480 BV510 BV570 BV605 BV650 BV711 BV750 BV785 BB515 AF488 AF532 PE PE/Dazzle594 PE-Cy5 PerCP-Cy5.5 PerCP-eF710 PE-Cy7 APC AF647 APC-R700 APC-Fire750 BV421 SB436 eF450 BV480 BV510 BV570 BV605 BV650 BV711 BV750 BV785 BB515 AF488 AF532 PE PE/Dazzle594 PE-Cy5 PerCP-Cy5.5 PerCP-eF710 PE-Cy7 APC AF647 APC-R700 APC-Fire750 23 100 200 300 400 500 600 700 Stain Index Stain Index Blue Laser Excitable Dyes 200 400 600 800 1000 1200 Stain Index Stain Index Red Laser Excitable Dyes 50 100 150 200 250 300 Stain Index Stain Index Violet Laser Excitable Dyes 200 400 600 800 1000 1200 1400 1600 Stain Index Stain Index Blue Laser Excitable Dyes

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 41

What is spillover?

Spillover occurs when more than

  • ne fluorophore is

detected in one detector

Conventional flow: APC spills into the AF700 detector Spectral cytometry: Both BV421 and BV510 are in the V2-V15 detectors

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 42

Visualizing Spillover

Conventional Cytometry

This plot is:

  • Undercompensated
  • BV711 is spilling into BV421

Compensation fixes spillover

Spectral cytometry

I’m on the fence about using “undercompensated” and “BV711 spilling into BV421” to describe this, but no other terms exist

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 43

What is Spillover-Spreading?

  • Spillover-spreading (SS) occurs after compensation (and unmixing?)
  • SS is variance that occurs from errors in measurement/data binning and photon counting

statistics

  • There is measurement error when other fluorophores emit light in the same detector due to

spectral overlap

  • Photons are hard to count, there is error in counting them
  • (If you’re still confused there’s some further reading on the next slide)
PE-Cy7-A :: CD11c APC-Cy7-A :: CD45 Comp-PE-Cy7-A :: CD11c Comp-APC-Cy7-A :: CD45 PE-Cy7 APC-Cy7

Uncompensated Compensated: Ideal Compensated: Reality

Spillover- spreading

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 44

Compensation does NOT introduce or increase error (spreading), it only reveals it!

  • The measurement error is already present. Compensation does

not increase this error, it does not change it, it does not introduce any more error.

  • Compensation simply makes the error more apparent by

shifting it to the low end of the logscale.

  • Staining controls are necessary to define gate placement!
  • More reading on spillover-spreading:
  • Roederer M. Compensation in flow cytometry. Curr Protoc Cytom. 2002 Dec; Chapter 1:Unit 1.14. doi: 10.1002/0471142956.cy0114s22.
  • Nguyen R, et al. Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design. Cytometry A. 2013

Mar;83(3):306-15. doi: 10.1002/cyto.a.22251.

  • Roederer M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry. 2001 Nov 1;45(3):194-205.

Slide modified from M. Roederer: https://newenglandcytometry.files.wordpress.com/2011/06/mroederer.pdf Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 45

Why do we need to worry about spillover-spreading?

  • The more colors in a panel, the more spillover-spreading occurs
  • This happens with both conventional and spectral cytometry
  • Spillover spreading can decrease resolution of a positive

population

BV785 Single Stain Control Full stain (14 colors)

BV785 Single Stain Control Full Stain Unstained

Spreading leads to loss in resolution of positive population

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 46

What do we know about spillover-spreading?

  • More spreading occurs with brighter intensity
  • More spreading occurs with more fluorophores in the detector
  • More spreading occurs the more a fluorophore spills into another detector

Nguyen R, et al. Cytometry A. 2013 Mar;83(3):306-15. doi: 10.1002/cyto.a.22251.

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 47

More spreading occurs the more a fluorophore spills into another detector (Example: 16-color conventional cytometry)

PerCP-Cy5.5 spill into BUV737: 14 BUV737 spill into PerCP-Cy5.5: 13.3

PerCP-Cy5.5 CD66b BUV737 CD8

Higher compensation values, bright intensity (y axis), more spreading

BUV737 PerCP-Cy5.5

PerCP-Cy5.5 CD66b BUV805 CD3

PerCP-Cy5.5 spill into BUV805: 1.1 BUV805 spill into PerCP-Cy5.5: 0

Low compensation values, medium intensity, no spreading

BUV805 PerCP-Cy5.5

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 48

How do we minimize spillover-spreading?

  • Better panel design!
  • Use dim markers for fluorophores that cause lots of spreading
  • Plan your panel so that fluorophores that spread into each other are on

different cell types

PerCP-Cy5.5 CD66b BUV737 CD8

A+B+ cells A+B- cells A-B+ cells PerCP/Cy5.5 anti-A BV650 anti-B PerCP/Cy5.5 anti-A BV711 anti-B A-B+ cells A+B+ cells A+B- cells Diagrammatic example:
  • PerCP/Cy5.5 (with marker “A”) spreads into the BV711 and BV650 detectors, leading to a loss of resolution in both.
  • Figure 1: BV711 is bright enough, and marker “B” is expressed highly enough that A+B+ cells can be resolved from A+B- cells.
  • Figure 2: However, BV650 is not bright enough to resolve A+B+ cells from A+B- cells.
Figure 1: resolvable Figure 2: not resolvable Spreading error is what causes problems for resolving true positive populations due to spreading of the negative population. The process of correct compensation reveals (not creates) measurement error, known as spreading error (SE)

CD66b+CD8+ should never exist! No need to worry about spreading error

Cytometry and Antibody Technology Facility Aurora Training Course

slide-49
SLIDE 49

Understanding the spread matrix for Aurora panel design

  • This table was created with CD4 reference controls for all

fluorophores

  • This table assumes all markers are bright and all markers are in the

tube

To read this table: spread of fluor in the row impacts resolution of the fluor in the column. Red means the fluor in that row has significant spread into the dye in the column (for example PE into AF532). Areas in bright pink and

BV421 SB436 eF450 BV480 BV510 BV570 BV605 BV650 BV711 BV750 BV785 BB515 AF488 AF532 PE PE/Dazzle594 PE-Cy5 PerCP-Cy5.5 PerCP-eF710 PE-Cy7 APC AF647 APC-R700 APC-Fire750 BV421 SB436 eF450 BV480 BV510 BV570 BV605 BV650 BV711 BV750 BV785 BB515 AF488 AF532 PE PE/Dazzle594 PE-Cy5 PerCP-Cy5.5 PerCP-eF710 PE-Cy7 APC AF647 APC-R700 APC-Fire750

Cytometry and Antibody Technology Facility Aurora Training Course

slide-50
SLIDE 50

Fluorochrome assignment

  • Fluorochrome assignment for tertiary antigens
  • Assess reagent availability (often not too many options available)
  • Assign brighter fluorophores (use fluorophore brightness ranking)
  • Fluorochrome assignment for secondary antigens
  • Based on CO-EXPRESSION of antigens expressed at intermediate levels
  • If no co-expression, use any bright dye still available
  • If co-expression:
  • if available, use a bright dye that does not spread into selected fluor for tertiary antigens
  • If only available dyes have spread, use a dim dye to minimize spread impact
  • Fluorochrome assignment for primary antigens
  • Often available in many colors
  • Try to assign dyes that are dim and that have minimal spread into other dyes. Primary dyes can

tolerate many other fluorophores impacting their spread (Ex: PerCP-Cy5.5, AF700, AF532, eFluor450, BV510)

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 51

Live/Dead Dyes

  • Cytek recommends BioLegend’s Zombie NIR
  • There may be issues with AF700 … TBD
  • Best live/dead reference control: Kill some cells, stain with

Zombie NIR, wash, right before running the control spike in some unstained cells

  • Many ways to kill cells – I like 65°C for 10 minutes
  • DAPI and PI can be use for live/dead – make sure you titrate!!!

Cytometry and Antibody Technology Facility Aurora Training Course

slide-52
SLIDE 52

Titrations and Staining Conditions

Cytometry and Antibody Technology Facility Aurora Training Course

slide-53
SLIDE 53

All antibodies should be titrated before your experiment

43175

APC-CD24 SSC-A

69170

APC-CD24 SSC-A

74930

APC-CD24 SSC-A

2 uL 0.2 uL 0.6 uL Amount recommended by Manufacturer

  • Antibody titration becomes increasingly important with larger panels

Amount used for expt

Cytometry and Antibody Technology Facility Aurora Training Course

slide-54
SLIDE 54

Non-specific binding lowers resolution

2 uL 6 uL 0.2 uL 0.6 uL Amount used for expt

Cytometry and Antibody Technology Facility Aurora Training Course

slide-55
SLIDE 55

Can you use antibodies that were titrated on a fortessa?

  • Probably. Make sure you test the antibody on the aurora first to make sure

the fluorophore is too bright

  • The most common issue that I have seen is the live dead dyes – we

recommend you titrate those on the aurora

Zombie NIR-A :: live dead SSC-A Zombie NIR-A :: live dead SSC-A Zombie NIR-A :: live dead SSC-A Zombie NIR-A :: live dead SSC-A

0.3 uL 1 uL 0.03 uL 0.1 uL Too bright and off scale! Too bright and off scale!

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 56

Staining Conditions: Impact of Washes on BB515 Background

25

CD8 BB515 50 100 200 400 800 1600 3200 6400

titration

Wash #1 Wash #2 Wash #3 0.3*106 resuspended in 150ul Run on Aurora 0.3*106 resuspended in 150ul Run on Aurora 0.3*106 resuspended in 150ul Run on Aurora mouse splenocytes FcBlock 1:50 10 min on ice, then plated 106 / well Stained in FACS buffer 40 min incubation

  • n ice

Anna Belkina, MD PhD @ FCCF Boston University

Cytometry and Antibody Technology Facility Aurora Training Course

Slides from Cytek an Anna Belkina

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SLIDE 57

Staining Conditions: Impact of Washes on FITC

CD8 BB515 50 100 200 400 800 1600 3200 6400

Titration

Wash #1 Wash #2 0.3*106 Resuspended in 150ul Run on Aurora 0.3*106 Resuspended in 150ul Run on Aurora

50 400 50 400 50 400

Mouse splenocytes FcBlock 1:50 10 min on ice, then plated 106 / well Stained in FACS buffer 40 min incubation

  • n ice

Anna Belkina, MD PhD @ FCCF Boston University

26

Wash #3 0.3*106 Resuspended in 150ul Run on Aurora Cytometry and Antibody Technology Facility Aurora Training Course

Slides from Cytek an Anna Belkina

slide-58
SLIDE 58

Need or No Need for Polymer Stain Buffer

27

WITH Brilliant Stain Buffer No comp corrections applied WITHOUT Stain Buffer WITH Super Bright Stain Buffer No comp corrections applied

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 59

Does Optimal Titration Prevent the Need for Staining Buffer?

28

WITH Brilliant Stain Buffer No comp corrections applied WITH Brilliant Stain Buffer All antibodies 10x lower concentration WITHOUT Brilliant Stain Buffer All antibodies 10x lower concentration

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 60

Other considerations for staining

  • If you have a very large panel and larger antibody volumes

(5 µL/test), you may have more than 100 µL of antibodies

  • In this case, you may need to increase your staining volume to

150-200 µL

  • Beware of aggregates in your antibodies
  • Centrifuge your antibodies 3 minutes at 10,000 RPM before using

Aggregates: Keep an Eye on Them!

29

Anna Belkina, MD PhD @ FCCF Boston University

Experiment 1: CCR7 BV480 lot 1 CCR7 FMO CCR7 lot 9092880 CCR7 lot 9092880 new vial CCR7 lot 9092880

  • ld vial

CCR7 lot 9007827 new lot, new vial Experiment 2: CCR7 BV480 vials were spun down for 3 min at 10,000 rpm An additional vial of same lot and a new vial of a different lot were provided by the manufacturer (BD Biosciences) for troubleshooting

Aggregates: Keep an Eye on Them!

29

Anna Belkina, MD PhD @ FCCF Boston University

Experiment 1: CCR7 BV480 lot 1 CCR7 FMO CCR7 lot 9092880 CCR7 lot 9092880 new vial CCR7 lot 9092880

  • ld vial

CCR7 lot 9007827 new lot, new vial Experiment 2: CCR7 BV480 vials were spun down for 3 min at 10,000 rpm An additional vial of same lot and a new vial of a different lot were provided by the manufacturer (BD Biosciences) for troubleshooting

After centrifuging: Before centrifuging:

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 61

Workflow for using the SpectroFlo software on the aurora

  • 1. Run unstained cells
  • 2. Preview a fully stained sample to check if any gains need to

be lowered

  • 3. Record reference controls
  • 4. Spectral unmixing (recommended but optional at this step)
  • 5. Run full stained samples
  • 6. Analyze samples in FlowJo or FCS Express
  • 7. (Spectral unmixing can be done again if needed)

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 62

Cytek Assay Settings

  • One of the easiest things about the aurora is that you don’t

usually need to adjust the gains (voltages) for every detector

  • The the Cytek Assay Settings and daily QC automatically set

the gains for you!

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 63

What are the cytek assay settings?

  • The term “cytek assay settings” has been used to refer to two

things:

Spectra Differences in Bead Lots

Bead lot 2002 has better signal at longer wavelengths especially from UV and violet laser excitation:

9

Bead lot 2002 Bead lot 1001

Required for UV laser Current bead lot as of 07- 2019 MFIs of the QC beads: The gains set on the instrument:

Cytometry and Antibody Technology Facility Aurora Training Course

slide-64
SLIDE 64

What are the cytek assay settings?

  • At cytek headquarters, cells were stained with individual

fluorophores and they determined the best gains for each detector to get each fluorophore to have a distinct signature and minimize spreading errors

  • QbSure beads (6 peaks of fluorescent intensity) were run at

those optimized gains to determine target MFIs for each detector

  • The cytek assay settings on each machine are essentially the

target MFIs determined at cytek HQ on their one specific aurora

Cytometry and Antibody Technology Facility Aurora Training Course

slide-65
SLIDE 65

What does daily QC do?

  • Every day when daily QC is run, gains are automatically

adjusted so that the positive peak of the QC beads match the target MFIs (the cytek assay settings)

  • Therefore, every time QC is run, the gains will change slightly
  • This works because the gains and the MFI are directly

proportional:

  • If the MFI is 500, the gain is 1000 and you want to decrease the MFI to

250, then the gain should be set at 500.

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 66

Daily QC: diluting the QC beads

To achieve best FSC CV, diluent of the beads must match the type of sheath used in the instrument Differences in diluent result in larger FSC CVs and can lead to fail QC that does not reflect instrument performance

FSC %rCV Difference In Sheath vs DI Water Diluted Beads

13

FSC %rCV = 2.25 FSC %rCV = 4.44

Sheath DI Water

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 67

Daily QC: re-using beads that were previously diluted

  • Diluted QC beads should only be re-used if they are stored in the fridge

protected from light!

  • If stored properly, diluted QC beads are stable for 5 days

5 Laser Aurora QC Bead Stability

12

REFERENCE BEADS STORED SHEATH DILUTED BEADS STORED DI DILUTED BEADS

MFI %rCV

Data from Cytek (Fresh)

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 68

So what do I need to do when I start my experiment?

  • 1. Run daily QC
  • Dilute beads in 1X PBS
  • Only reuse diluted beads if stored in the fridge
  • 2. Open your experiment and check that the assay settings are

set to “CytekAssaySettings NEW 0719”

  • No one will be able to save over these settings! Any user who

accidentally saves will be forced to save as

  • 3. Change your FSC and SSC as needed for your specific cells,

but do not change any gains on the lasers without first talking to David or Laura

Cytometry and Antibody Technology Facility Aurora Training Course

slide-69
SLIDE 69

What should you do at the end of your experiment?

  • Please help us keep the aurora clean by running the fluidics

shutdown when you have completed your experiment

  • Follow the on-screen instructions with 10% contrad followed by water
  • Please make sure that a tube of water is left on the aurora when you

leave

There is a handy sign on the front of the aurora to help you fill the tubes easily!!!

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 70

Performing Spectral Unmixing

Cytometry and Antibody Technology Facility Aurora Training Course

slide-71
SLIDE 71

Cytometry and Antibody Technology Facility Aurora Training Course

slide-72
SLIDE 72

Tips for Gating Reference Controls

Tips for Gating Reference Controls

48

Tips for Gating Reference Controls

48

Tips for Gating Reference Controls

48

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-73
SLIDE 73

Antigen Level of Expression and Spectrum

0.00% 5.00% 10.00% 15.00% 20.00% 25.00% 30.00% CD56 BV650 +++ CD56 BV650 ++ CD56 BV650+

Median V10-A CD56 BV650 +++ 17.33% CD56 BV650 ++ 18.13% CD56 BV650+ 20.03%

  • 0.45%
  • 1.31%
  • 4.12%

44

CD56+++ BV650 CD56++ BV650 CD56+ BV650 Spectrum Overlay Unmixed Data, BV605 vs SB600

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-74
SLIDE 74

Evaluation of Autofluorescence

Cytometry and Antibody Technology Facility Aurora Training Course

slide-75
SLIDE 75

High vs Low Autofluorescence (Non-UV Laser Configurations)

31

Splenocytes Lung Cells Yeast Cells

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-76
SLIDE 76

Extracting Low Autofluorescence (Non-UV Systems)

32

Autofluorescence Signature Whole Blood NO AF extraction WITH AF extraction

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-77
SLIDE 77

When should autofluorescence extraction be used?

  • Only use the autofluorescence extraction on cells with high

autofluorescence

  • On cells with low autofluorescence, the extraction can make

your data look worse

  • How do you determine what the cutoff is for high vs low

autofluorescence? TBD…

Cytometry and Antibody Technology Facility Aurora Training Course

slide-78
SLIDE 78

Check Unmixing Accuracy

Cytometry and Antibody Technology Facility Aurora Training Course

slide-79
SLIDE 79

Checking Unmixing Accuracy of Single Stained Cells

BV711 vs all other fluorochromes Alexa Fluor 488 vs all other fluorochromes

49

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-80
SLIDE 80

The Negative Populations Say It All….

32

These kinds of patterns tell me the unmixing worked AND panel design both worked well These kinds of patterns tell me the unmixing worked but there are some spread issues due to panel design

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-81
SLIDE 81

The Negative Populations Say It All….

33

All these different patterns show that the unmixing has errors… have you seen any of these in your data?

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-82
SLIDE 82

Unmixing errors can often the cause of weird looking negative populations

Finding Unmixing Errors Often Fixes Negative Populations

34

Slides from Cytek

Cytometry and Antibody Technology Facility Aurora Training Course

slide-83
SLIDE 83

Confused about your data?

  • Set up a meeting to talk to Laura!
  • LJohnston@bsd.uchicago.edu
  • I can talk to Monica DeLay (Cytek Technical Application

Specialist) or you can also email her!

  • mdelay@cytekbio.com

Cytometry and Antibody Technology Facility Aurora Training Course

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SLIDE 84

Other Notes

  • Next step: 1 hour hands on training with compensation beads
  • Monthly aurora user group meeting – second Friday of every

month

  • Mailing list – Aurorausers@lists.uchicago.edu

Cytometry and Antibody Technology Facility Aurora Training Course