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Polychromatic Roadshow
Wellington : Perth : Brisbane : Sydney : Melbourne
Reagents
Instrument Setup, Optimization, and QC
UV Brilliant UV, Quantum Dots, Vio BD (4), Invitrogen (8), Miltenyi (1)
Violet Brilliant Violet, Horizon, Pacific, BD (8), BioLegend (3), Coulter (2), Invitrogen (8), Miltenyi (2)
Quantum Dots, Vio
Blue Brilliant Blue, FITC, Alexa 488, BD (3), BD, Coulter, BioLegend, Invitrogen, Miltenyi (2)
Cy55PerCP, PE and Tandems, Vio
Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi
Red APC and Tandems , Alexa BD, Coulter, BioLegend, Invitrogen, Miltenyi
Equipment Decisions : Laser Choice
UV Brilliant UV, Quantum Dots, Vio BD (4), Invitrogen (8), Miltenyi (1)
Violet Brilliant Violet, Horizon, Pacific, BD (8), BioLegend (3), Coulter (2), Invitrogen (8), Miltenyi (2)
Quantum Dots, Vio
Blue Brilliant Blue, FITC, Alexa 488, BD (3), BD, Coulter, BioLegend, Invitrogen, Miltenyi (2)
Cy55PerCP, PE and Tandems, Vio
Green PE and Tandems, Vio BD, Coulter, BioLegend, Invitrogen, Miltenyi
Red APC and Tandems , Alexa BD, Coulter, BioLegend, Invitrogen, Miltenyi
Proprietary dyes,
Somewhat limited dye selection for multiplexing,
Catalogue of antibodies somewhat limited.
Keep reagent availability in mind for laser decisions.
UV or Violet Laser? Our System
(15 colors/ 2 lasers)
UV Dye V Dye
Well, if you can, do both. BUV390 BV421
Reagents will become available. UV450 (LD) BV510
This maximizes multiplexing. BUV490 BV570
BUV550 BV605
BUV680 BV650
BUV737 BV711
BUV800 BV750
BV785
Otherwise, if you have to choose one:
UV BUV390 UV450 BUV490 QD54 QD58 QD605 QD655 QD705
5 5 BV605 BV650 BV711
BV570
V BV421 V545 BV570 QD60 QD65 QD705 BV750 QD800
BV510 5 5 BV711 BV785
BV605 BV650
Blue or Green?
UV Violet Blue
CD4 BUV395
CD4 BV650
CD4 BB515
Laser Power (mW) Laser Power (mW) Laser Power (mW X 102)
Green Red
CD4 APC
Stain cells with
CD4 PE
representative conjugate
for each laser.
UV Violet Blue
CD4 BUV395
CD4 BV650
CD4 BB515
Laser Power (mW) Laser Power (mW) Laser Power (mW X 102)
Green Red
CD4 APC
Run samples at different
CD4 PE
laser powers.
Requires tunable lasers.
UV Violet Blue
CD4 BUV395
CD4 BV650
CD4 BB515
Laser Power (mW) Laser Power (mW) Laser Power (mW X 102)
Green Red
CD4 APC
CD4 PE
30 100 250
500 500
Filter-related Check that they didnt have a bad day at the factory.
Reference:
Perfetto, et al. Quality Assurance for Polychromatic Flow Cytometry,
Nature Protocols (2006 and 2012, in press).
Verifying Signals
Filter-related Check that they didnt have a bad day at the factory.
Reference:
Perfetto, et al. Quality Assurance for Polychromatic Flow Cytometry,
Nature Protocols (2006 and 2012, in press).
Cascade Test
1. All PMT @ 500 volts.
2. Remove all filters, reserve
last set for test.
1 3. Place test filter at first
PMT.
4. Run 1x beads.
5. Record CV.
6. Move test filters to next
4 3 PMT.
7. Return first PMTs
original filters.
8. Repeat for other PMTs.
9. Calculate statistical
photoelectron estimate =
2 1/n2, where n=CV/100.
Filter-related Check that they didnt have a bad day at the factory.
Reference:
Perfetto, et al. Quality Assurance for Polychromatic Flow Cytometry,
Nature Protocols (2006 and 2012, in press).
Troubleshooting Filter Issues
PMT 1 PMT 2 PMT 3
1x Rainbow Beads
3 All PMT @ 500 volts.
Case A
1 0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
Case B
2
2 3 4 5 2 3 4 5 2 3 4 5
A = OK! 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
Case C
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
Troubleshooting Filter Issues
PMT 1 PMT 2 PMT 3
1x Rainbow Beads
3 All PMT @ 500 volts.
Case A
1 0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
x
Case B
2
2 3 4 5 2 3 4 5 2 3 4 5
B = PMT2 Dichroic is 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
Troubleshooting Filter Issues
PMT 1 PMT 2 PMT 3
1x Rainbow Beads
3 All PMT @ 500 volts.
Case A
1 x 0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
0 10
2
10
3
10
4
10
5
x
Case B
2
2 3 4 5 2 3 4 5 2 3 4 5
C = PMT1 Dichroic is a 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5
Verifying Signals
Filter-related Check that they didnt have a bad day at the factory.
Reference:
Perfetto, et al. Quality Assurance for Polychromatic Flow Cytometry,
Nature Protocols (2006 and 2012, in press).
Instrument Setup, Optimization, and QC
Standardize Sample
Preparation and Handling
Approaches to
Optimization/Standardization To Date
Q and B Calculations
Bead-Based Protocols Using an LED Pulser
(2014 - )
Q & B: Parameters Defining Separation
Positive Gate
Limit of Detection
Negative Gate
Sources of Background
Q & B: Parameters Defining Separation
Positive Gate
Limit of Detection
Negative Gate
Sources of Background
10%
5%
2.5%
1.25%
Q & B: Parameters Defining Separation
PMT Quality
(Efficiency of Photon to Signal)
Electronic Auto- Spread Poor -------------------------- Good
Noise Fluor.
PMT Quality
(Efficiency of Photon to Signal)
Electronic Auto- Spread Poor -------------------------- Good
Noise Fluor.
B Q
Q & B Can Be Measured with LED Pulser
119 5174
Count
Count
79 3450
40 1725
0 0
2 1 2 3 4 5 2 1 2 3 4 5
-10 -10 10 10 10 10 -10-10 10 10 10 10
FITC-A FITC-A
Jim Wood, Wake Forest University and Steve Perfetto, VRC, NIH
Q & B Calculations
Set Reasonable
Voltage
Repeat Lower
Intensities (Dial)
Output = Q and B = rough because B is voltage dependent,
rough B Value and weve selected only one voltage here.
Why do this?
120
100
B-Values
B Value (rSD)
80
60
40
20
0
Aria-B Aria-C LSR-A LSR-B LSR-C LSR-D LSR-E pX50
Instrument
Instrument
Huge spread in B values across detectors and instruments.
This is why standardization of sample processing will never fully make data across
instruments comparable. For new instruments, we tried to lessen this issue.
Q & B Values on Detectors for
New VRC Instruments
780nm
B-Values
Q-Values
Q & B Values on Detectors for
New VRC Instruments
Exclude detectors with high
background and low 780nm
resolution.
Q-Values
So Far
Verified filters
Bead-based
Auto-fluorescence / B ratio
Wavelength-based
Bead-based Method for Voltages
1) Check that filters are in place; set all voltages to 350.
Auto-fluorescence to B
Pulser triggers Make sure Set voltage, Increase voltage,
SSC lasers on collect data collect data
Gains = 300v
Gains = 430v Gains = 500v
8000
4000
1500
6000
3000
# Cells
# Cells
# Cells
1000
4000
2000
2000 500
1000
0 0 0
0 10 2 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5 2 3 4 5
G560-A: Fixed Unst PBMC 0 10 10 10 10
G560-A: Fixed Unst PBMC G560-A: Fixed Unst PBMC
B Values at different Gains
Gains = 300v Gains = 430v Gains = 500v
Auto-fluorescence to B
Pulser triggers Make sure Set voltage, Increase voltage,
SSC lasers on collect data collect data
Gains = 300v
Gains = 430v Gains = 500v
8000
4000
1500
6000
3000
# Cells
# Cells
# Cells
1000
4000
2000
2000 500
1000
0 0 0
0 10 2 10 3 10 4 10 5 0 10
2
10
3
10
4
10
5 2 3 4 5
G560-A: Fixed Unst PBMC 0 10 10 10 10
G560-A: Fixed Unst PBMC G560-A: Fixed Unst PBMC
Mid-point is
preliminary voltage for
each detector.
Wavelength-based
= Optimized system,
easy voltage setting.
Clean-up Voltage Choices
To select best voltage for each detector, use Comp beads (IgK capture beads) to capture
antibodies labeled with the specific fluorochromes used in experiments. Run at 50 volt
increments, within range of voltages determined in first part of routine. Select voltage
for each detector where 1o signal for detector is higher than 2o signals.
Clean-up Voltage Choices
Pair this with a bright dye (BV421, PE) for SuperHiRes Channel.
Pulsed LED
(light detection)
Standard Beads
(known # FL molecules)
Calibration known # of
dye molecules per mAb
Steve Perfetto
Q and B to Predict Panel Success
Establish a tolerance range, based on 20+ runs. Calculate the CV for the multiple
runs, and make the target MFI +/- CV of 10% your QC limit.
2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5
0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10
Standardizing Daily Performance
Daily operation:
2.If not, then check instrument and beads first, then adjust
voltage (but be sure still within range that calibration routine
defined.
12
11 LSR-A
14
10
12
9
rCV G560
10
8
rCV G560
7 8
UCL=6.72
6 6
Avg=5.01
5 4
LCL=3.30
4 2
12 36 60 84 108 132 156
3
Time
2
LSR-A
12
11
600
10
9
550
8 New PE detector required lower
rCV G560
500 6
4
450
3
LSR-E LSR-E
Summary: What Did We Learn