Where to Flow? Challenges, Solutions & Innovations in modern Flow Cytometry 1st Pan Arab Flow Cytometry Working Group Workshop Friday, October 14th, 2016 Cairo, Egypt Dr. Gerald Pfister, PhD, CCy Manager Flow Cytometry Core Facility Qatar Biomedical Research Institute, Doha, Qatar Web: www.qbri.org.qa / E-mail: gpfi[email protected]
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Dr. gerald pfister challenges, solutions and innovations in modern flowcytometry lab
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Where to Flow?Challenges, Solutions & Innovations in modern Flow Cytometry
1st Pan Arab Flow Cytometry Working Group Workshop
Friday, October 14th, 2016
Cairo, Egypt
Dr. Gerald Pfister, PhD, CCyManager Flow Cytometry Core Facility
Mass Cytometry – the CyTOF2™, Fluidigm, Inc.Use of antibodies coupled to isotopically purified mass tagsQuantified in individual cells by an Inductively-Coupled Plasma Mass Spectrometer (ICP-MS)
About 40-simultaneous antigens can be quantified at a rate of about 500-1000 cells/second
Source: http://cytof.scilifelab.se/
What are the Challenges & Innovations in Flow Cytometry?
Example: Influence of number of events recorded on data accuracy
Increasing Variability of data
Number of events
Data Analysis – Statistics in Population Analysis
Quantification of the average intensity of a parameter in a population
M F Iean luorescence ntensity
Arithmetic mean ?Geometric mean ?
Median ? Mode ?
Data Analysis – Statistics in Population Analysis
Median Fluorescence Intensity is often the best choice for biological samples
Data Analysis – Statistics in Population Analysis
Coefficient of Variation (CV)
Data Analysis – Statistics in Population Analysis
Effect of high CV’s on resolving populations
Bad resolution
Good resolution
Data Analysis – What do we see on our Plots?
http://www.emdmillipore.com
Events (not cells!)
Data Analysis – Gating Strategy
1: Flow stability gatingTo capture events once the flow stream has stabilized, eliminating effects of clogging, back-
pressure, and other instrument issues
2: Pulse geometry gatingTo remove doublets from the dataset
3: Forward and side scatter gatingTo remove debris and other events of non-interest while preserving cells based on size and or complexity
4: Subsetting gatingTo rely on expression of markers and what they identify. Using viability dyes and dump
channels further narrow to the cells of interest. This is where Fluorescence Minus One (FMO) controls become critical in defining the populations of interest
5: BackgatingTo provide visualization of cells in final gate at higher level