Transcript No Slide Title

How to set up and run an LSRII
Holden T. Maecker
Outline
• Initial instrument characterization
• Periodic (weekly) QC
• Daily (experiment-specific) setup
Initial instrument characterization
• Exp 1: Run 8-peak beads
• Set voltages so that brightest bead is ~105
• Observe peak separation in various
detectors (need a standard)
• Can be used to check area scaling factor
(compare beads in Area versus Height for
one detector on each laser)
• Can be used to check laser delay (if
sensitivity is reduced in all detectors on
one laser)
What is laser delay?
Time from intersection
of 1st to 2nd laser
Time from intersection
of 1st to 3rd laser
• Dependent upon sheath velocity
• Can be mitigated by larger window extensions
What is a window extension?
Time window over which a pulse signal is measured
• Zero is the narrowest window, two is default
• Larger window extensions can overcome
minor imprecisions in laser delay setting
What is “area scaling factor”?
• A factor that is applied to the pulse area
measurement to equalize the height and area
scales
• Should never need to be adjusted
• Is relatively inconsequential to typical
immunophenotyping experiments
Initial instrument characterization
• Exp 2: Determine minimum baseline PMT voltages
• Run peak 2 beads at a series of voltages (350-800
volts)
• Export CV of tightly-gated singlet beads for each
detector
• Plot CV versus voltage for all detectors
• Lowest plateau voltage is the minimum baseline
voltage for that detector
• Run mid-range (peak 3) beads at minimum
baseline voltages, record mean in each detector
(these are your initial baseline target values)
Results of initial PMT voltage determination
Blue laser
P1 FITC-A CV
P1 PE-blue-A CV
P1 PE-Texas Red-A CV
P1 PerCP-Cy5-5-A CV
P1 PE-Cy7-A CV
CV
1000
100
10000
10
100
10
1
200 300 400 500 600 700 800 900 1000
1
200 300 400 500 600 700 800 900 1000
voltage
voltage
Red laser
10000
Violet laser
10000
P1 APC-A CV
P1 Alexa 700-A CV
P1 APC-Cy7C-A CV
100
10
P1 Pacific Blue-A CV
P1 AMCyan-A CV
P1 565 QDot-A CV
P1 605 QDot-A CV
P1 655 QDot-A CV
P1 705 QDot-A CV
1000
CV
1000
CV
P1 PE-green-A CV
P1 PE-Texas Red-green-A CV
P1 PerCP-Cy5-5-green-A CV
P1 PE-Cy7-green-A CV
1000
CV
10000
Green laser
100
10
1
200 300 400 500 600 700 800 900 1000
1
200 300 400 500 600 700 800 900 1000
voltage
voltage
Periodic (weekly) QC
• Open Exp 1 (8-peak beads)
• Run a new tube of 8-peak beads
• Compare histograms to initial run
• If only one detector has lost sensitivity:
• Check filters
• Check PMT (BD service)
• If all detectors have lost sensitivity:
• Check fluidics for bubbles, prime
• If all detectors on one laser have lost sensitivity:
• Check laser delay
• Check laser output (BD service)
• Consider tracking one peak (#6) from each detector
for MFI and CV over time
Daily (experiment-specific) setup
• For a new panel:
• Run mid-range beads and set voltages to achieve initial
baseline target values (or target values from a previous
panel)
• Run fully stained cells and reduce voltages in any detectors
where events are off-scale
• Run CompBeads (stained with panel-specific antibodies)
and calculate compensation
• Re-run fully stained cells and increase voltages where
negative populations are below zero, if desired
• If any voltages were changed, repeat compensation and
then check fully stained cells again
• Run mid-range beads (with compensation OFF) and record
new panel-specific target values
• Run samples (with compensation ON)
Daily (experiment-specific) setup
• For a panel with previously determined target values:
• Run mid-range beads and set voltages to achieve previously
determined target values
• Run CompBeads (stained with panel-specific antibodies)
and calculate compensation
• Run samples
Acknowledgements and resources
• Joe Trotter, BD Biosciences
• http://maeckerlab.typepad.com
• 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.