cell sorting using flow cytometry - aacc
TRANSCRIPT
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© Clinical Chemistry
Cell Sorting using Flow Cytometry
Michael Timm, BA
Development Coordinator, Department of
Laboratory Medicine and Pathology, Mayo
Clinic
10.15428/CCTC.2019.307215
01/02/2019
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What is Cell Sorting?
• A process of physically separating a cell population in
a suspension from the rest
size, charge
protein expression
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Types of Cell Sorting in the Routine
Use Today
• Magnetic Activated Cells Sorting (MACS)
• Magnetic nanoparticles bound to the antibody
• BULK = all cells collected at once (fast and
great for large quantities)
• Only one cell characteristic used
• Fluorescence Activated Cell Sorting (FACS)
• Fluorescent dyes bound to different antibodies
• SINGLE CELL = each cell separately analyzed
• Multiple characteristics used
• High specificity and purity
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History of Cell Sorting
Late 1800s
• Lord Rayleighs principle of droplet formation from a stream of liquid
Mid-1960’s
• Sweet develops first INK JET at Stanford University
• Fulwyler at Los Alamos National Laboratory first successful sort of cells based cell volume
Late 1960’s
• Len Herzenberg coined the commonly used term FACS –Fluorescence Activated Cell Sorter
Early 1970s
• Becton Dickinson launched first commercially available cell sorter the FACS-1. BD still owns the trademark for FACS to this day
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FACS vs. Flow Cytometry
• All the principles of flow cytometry immunophenotyping
apply to FACS
• Fluidics
• Optics
• Electronics
• FACS has an additional component of cell collection,
defined by gating
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FLOW
CYTOMETRY
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SAMPLE
SHEATH FLUID
HYDRODYNAMIC
FOCUSING
WASTE
DIGITAL OUTPUT
(DOT PLOTS)LASER
LIGHT
SCATTER
FLUORESCENCE
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FACS
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LASERLIGHT
SCATTER
FLUORESCENCE
SAMPLE
SHEATH FLUID
HYDRODYNAMIC
FOCUSING
WASTE
CHARGE
-+
SORTED fraction 2PHYSICAL OUTPUT
(CELLS)
DIGITAL OUTPUT
(DOT PLOTS)
DROP
DELAY
SORTED fraction 1
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FACS Experiment
Molecular
analysis
Sorting criteria Purity assessment Downstream assay
Molecular
analysis
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Applications of Cell Sorting
• Subset analysis of any lineage
• Increased sensitivity and specificity of genetic
testing (PCR, FISH, NGS, microarrays…etc)
• Isolation of stem cells
• Isolation of engineered cells
• Cloning (single cell sorting)
• Protein engineering
• Drug discovery
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Evolution of Cell Sorting
EARLY FACS MODERN FACS
INSTRUMENT SIZE LARGE SMALL
EASE OF USE DIFFICULT EASY
NUMBER OF
FLUOROCHROMES2 UP TO 9
NUMBER OF
STREAMS
(COLLECTION
OUTPUTS)
1 UP TO 4
SPEED SLOW FAST
COLLECTING
TUBES
LIMITED
OPTIONSMANY OPTIONS
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Choosing the Right Instrument
– Workflow Considerations
• COMPLEXITY - number of sort streams for recovery
• 1 or 2 – small benchtop models
• More than 2 – large floor models
• NEED FOR LIVE CELLS (culturing, cloning)
• Sterile sorts
• SPACE AND COST
• Floor models are larger and much more expensive
• AEROSOLIZATION
• Need a hood or integrated aerosol management
system
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Optimizing and Troubleshooting a
Sorting Assay• Depends on the DOWNSTREAM ASSAY
• Cell number, purity, viability, fixation
• Cell aggregation can be a problem
• Filtering, diluting, adding EDTA or DNAse
• Instrument set up
• PMTs and compensation
• Collection
• May need a specific buffer, or specific collection tubes
• Flow rate is inversely proportional to the purity of the target
population
• Purity vs. recovery mode
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References
1. Orfao A, Ruiz-Arguelles. General concepts about cell sorting techniques. Clincial Biochemistry 1996;29:5-9
2. American Society for Clinical Pathology, Flow Cytometry in Clinical Diagnosis (4th edtition). 2007:15-22
3. Dangl J, Lanier L. Founding father of FACS: Professor Leonard A. Herzenberg. PNAS 2013;110:20848-9
4. Hrezenberg LA, Parks D, Sahaf B, Perez O et al. The history and future of the fluorescence activated cell sorter and flow cytometry: A view from Stanford. Clinical Chemistry 2002;48:1819-27
5. https://bitesizebio.com/13693/historical-background-of-flow-cytometry/
Please provide full citations for your references in style with Journal format as explained here:
http://www.clinchem.org/site/info_ar/info_authors.xhtml#References
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Disclosures/Potential Conflicts of Interest
Upon Pearl submission, the presenter completed the Clinical Chemistry
disclosure form. Disclosures and/or potential conflicts of interest:
▪ Employment or Leadership: None declared
▪ Consultant or Advisory Role: None declared
▪ Stock Ownership: None declared
▪ Honoraria: None declared
▪ Research Funding: None declared
▪ Expert Testimony: None declared
▪ Patents: None declared
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