Odyssea of a Beotian in the world of avalanche photo-diodes (APDs)
Dr. Hervé Luche
Monday, July 24, 2017
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Chapters
Introduction
Mutiple steps for multicolor panel design tailored to CytoFLEX LX
A Unique Technology Institue For academia and industrial partners
All class of cytometers Optimal instrument for each type of assay
Our main activity
CytoFLEX PARTICULARITIES
What is so special about CytoFLEX LX?
Avalanche photo-diodes (APDs)
Wavelength-division multiplexing (WDM) - Wikipedia
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-phenogyping module: Flow, mass and spectral cytometrists
INSTRUMENT CONFIGURATION
CytoFLEX LX resolution using 8 peak rainbow beads
Laser order: why to bother?
UV Laser Off
Checking APD signal linearities
Linearity evaluation: Ratio 3/7 peak of Rainbow beads, normalize to 100% on minimal gain
Linearity range on BV605 channel
Dim and High signals visualized at the same time
Voltration on calibrite beads and unstained cells to determine minimal gain
Determine Optimal gain using SI
One major observation on CytoFLEX LX
Rank dyes according to their brightness on your instrument
Dyes tested to establish Brightness Index
Dyes not included in this ranking
Brightness Index on CytoFLEX LX
20 COLORS IMMUNOPHENOTYPING
How to select optimal antibody/fluorochrome pair?
Spillover spreading matrix (SSM) of CytoFLEX LX
Think at first the populations to be reported
Taint your cells with dyes with low spectral overlap
20-color panel to identify 33 populations of murine splenocytes
Compensation matrix
Myeloid cells
DCs/B-cells
T-cells
VOLUMETRIC COUNTING
CytoFlex Size Linearity 5 µm to 10 µm
Optimization FSC/SSC gain for absolute counting
Gating strategy for cell counting
Q/A for cell counting
Compare two automates for volumetric counting
FLUORESCENT BARCODING
Other application: quantify molecules on several background of mice at the same time
Quantification on rare cell population
Quantify markers upon thymic DC maturation
WIP: Automated analysis pipeline for antibody titration
Test FBC combinations with super bright dyes on CytoFLEX LX
Maximize dim and high resolution of FBC schemes
Barcoding scheme used in multicolor application
Example of data output obtained on CytoFLEX LX
Conclusion
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