Post on 08-Apr-2017
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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
Qatar Biomedical Research Institute, Doha, Qatar
Web: www.qbri.org.qa / E-mail: gpfister@qf.org.qa
The Qatar Biomedical Research Institute QBRI
http://www.qbri.org.qa
The Qatar Biomedical Research Institute QBRI
http://www.qbri.org.qa
What is Flow Cytometry?
From: www.keokimed.com
What is Flow Cytometry good for?
http://www.accessexcellence.org/RC/VL/GG/
2015
15000
Why is Flow Cytometry important?
Published papers containing the key word „Flow Cytometry“
What are the Challenges & Innovations in Flow Cytometry?
LaboratoryInstrumentationHardware & SoftwarePerformance & QC
ApplicationsCell CultureCell Preparation & CollectionBiosafety
Data AnalysisData Management & StorageInterpretation of ResultsEducation & DevelopmentLab & User ManagementTroubleshooting
What are the Challenges & Innovations in Flow Cytometry?
LaboratoryInstrumentationHardware & SoftwarePerformance & QC
What Instrumentation is available and needed?
Basic
High-End
What Instrumentation is available and needed?
$
$$$
Instrumentation – Lasers & Optics
Instrumentation – Lasers & Optics
From the webinar „Fluorescent Proteins in Flow Cytometry“, June 26th 2015© CYTO University, by Teresa Hawley & Bill Telford
Instrumentation – Optimization, Performance Tracking & QC
Optimization of PMT-V enhances resolution of signals
T cells:SSClow / CD45high / SSClow / CD3high
B cells:SSClow / CD45high / CD3neg / CD19high
Monocytes:SSC / CD45interm / CD3neg / CD14high
PMT-V PE: 350V 400V 450V 500V550V
CD4 PE (0.1 ml / test)
Instrumentation – Recent Technologies
The ImageStreamX Imaging Flow Cytometer from Amnis / MilliporeCombines flow cytometry with microscopy for multiple applications
Source: http://www.amnis.com
Instrumentation – Recent Technologies
The ImageStreamX Imaging Flow Cytometer from Amnis / MilliporeMorphology: Shape change in primary monocytes
Source: http://www.amnis.com
Chemoattractant
Migration to sites
of inflammation
Reduced inflammatory response Autoimmune drugs
Instrumentation – Recent Technologies
Sorting examples: BD FACSAria FusionTM with Class II Biosafety Cabinet
Instrumentation – Recent Technologies
BD FACSAriaTM Fusion:Sorting of 100-160-200-240 nm beads (nanoparticles)
Scatter thresholding - 4-way sort
BioCytex Gigamix beads (Mixture of Megamix-Plus SSC and Megamix-Plus FSC) were prepared according to manufacturer’s instruction.
240 nm200 nm
160 nm
PRE-SORT
POST-SORT
530/30-H
SSC
-H
100 nm
900 nm
500 nm300 nm
240 nm200 nm160 nm
100 nm
Instrumentation – Recent Technologies
Small Particles: Analysis and Sort of Extracellular Vesicles (<300nm) on the BD InfluxTM
Instrumentation – Recent Technologies
The Sony SP6800 Spectral AnalyzerCollects all 500-800nm emitted light from sample and focus it onto 32-channel PMT
No optical filters and no conventional compensation needed
Up to 15 colors with two lasers, allows for use of high-spectral-overlap-dyes in one sample
Instrumentation – Recent Technologies
Analysis of marine water samples by the Sony SP6800 Spectral Analyzer
Source: https://precym.mio.univ-amu.fr/spip.php?article232
Instrumentation – Recent Technologies
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?
Cell CultureCell Preparation & CollectionApplicationsBiosafety
Cell Preparation & Collection
Sample quality & cell viability
Sample concentration
Fluorochrome selection
Cell Preparation & Collection
Sample quality & cell viabilitySample concentration
Fluorochrome selection
G I G Oarbage arbagen ut
Filter to avoid aggregates
Treat cells gently Check cell viability
dead
alive
Cell Preparation & Collection
Sample quality & cell viability
Sample concentration
Fluorochrome selection
• Instrument ConfigurationSelect fluorochromes according to optical configuration
• Fluorochrome brightnessPut bright fluorochromes to dim markers and vice versa
• Spectral SpilloverAvoid high spillover from markers on same cells
• Stability of tandem dyesDegradation due to light, fixatives, temperature, pH…
• Non-specific bindingConsider unspecific binding to FC receptors
• Size of fluorochromeHardly influences intracellular & extracellular staining
Applications – Sort for Single Cell RNA Sequencing
Single cell isolation techniques
Applications – Sort for Single Cell RNA Sequencing
Direct sort onto the PCR plate by the Fuidigm C1 system
Single Cell Transcriptomics, etc...
Advantages of Microfluidics:
AutomationHigh ThroughputLower risk of contamination
Indi
vidu
al c
ells
Individual genes
© Dr .Vasiliki Chini, QBRI
Biosafety – The Two Fundamentals
Risk ManagementProtective instrumentation and engineering controls
Personal protective equipment and clothing (PPE)
Safe laboratory procedures (SOP)
Laboratory design for containment
Risk AssessmentIdentification of hazards
Measurement of the risk that something will happen as a result of that risk
Takes into account the Risk Group and procedures performed
Biosafety – General Rules for Laboratory Work
European Directive 2000/54/ECNo eating and drinking in a lab
No mouth pipetting
Appropriate cleaning procedures
Lab design (floor, tables, cupboards)
Good laboratory practice (GLP) & standard operation protocols (SOP)
What are the Challenges in Innovative Flow Cytometry?
Data AnalysisData Management & StorageInterpretation of ResultsEducation & DevelopmentLab & User ManagementTroubleshooting
Data Analysis
Example: Influence of sample size and plot type on data display
Source: Practical Flow Cytometry 4th Edition, HM Shapiro, Wiley-Liss Press
Data Analysis
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
http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp
Data Analysis – Gating Strategy
Flow stability gating
http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp
Data Analysis – Gating Strategy
Pulse geometry gating (Doublet exclusion)
http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp
Data Analysis – Gating Strategy
Forward and side scatter gating
http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp
Data Analysis – Gating Strategy
Subsetting gating
http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp
Data Analysis – Gating Strategy
Backgating
http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp
Flow Resources: Possibilities, Possibilities, Possibilities...
Questions?
E-mail: gpfister@qf.org.qa