Speed Up Your Setup:
The CytoFLEX Platform Fast Track of 20-Color Panel Design
Dr. Hervé Luche
Thursday, July 5, 2018
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Chapters
Introduction
A Unique Technology Institute
All class of cytometers
High content immune phenotyping panels
Our main activity: Phenotyping mutant mice
CytoFLEX Particularities
What is so special about CytoFLEX LX?
Avalanche photo-diodes (APDs)
Wavelength-division multiplexing (WDM)
WDM on CytoFLEX
Specifications of CytoFLEX LX instrument for evaluation
Evaluation Program
Keys for success in multicolor panel design
CytoFLEX LX evaluation program
The Immuno-phenotyping module
Instrument Configuration
CytoFLEX LX resolution using 8 peak rainbow beads
Laser order: Why the bother?
NUV Laser Off
Violet laser off
Yellow/Green laser off
Red laser off
Checking APD signal linearities
Different measurements done one APDs using Rainbow beads
Linearity evaluation
Normalise to 100% on optimal gain and voltage range
Linearity range on BV605 channel
Dim and bright signals visualized at the same time
One major observation on CytoFLEX LX
Importance of dynamic linearity range
Multicolor Panel Design
How to establish high content multicolor panels
About dye brightness
Rank dyes according to their brightness on your instrument
Experimental workflow to establish a Brightness Index
Determine Optimal gain using SI
Dyes tested to establish Brightness Index
Dyes not included in this ranking
Brightness Index on CytoFLEX LX
20 Colors Immunophenotyping
Spillover Spreading Matrix (SSM)
Spectral overlap leading to data spreading is independant of compensation
Think at first the populations to be reported
Spillover spreading matrix (SSM) of CytoFLEX LX
Taint your cells with dyes with low spectral overlap
20-color panel to identify 33 populations of murine splenocytes
Myeloid cells
DCs/B-cells
T-cells
Volumetric Counting
CytoFlex Size Linearity 5 µm to 10 µm
Q/A for cell counting
Advantage of peristaltic pump over syringe pump for volumetric counting
Optimization FSC/SSC gain for absolute counting
Gating strategy for cell counting
Comparison of two cytometers for volumetric counting
No carry over between tubes
Conclusion
Acknowledgements
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