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Flow Cytometry

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

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Enrichment during transdominant genetic experiments using a flow sorter.

R Sandrock, J Karpilow, B Richards

|October 9, 2001

Summary

Flow cytometry with expression libraries enables genetic screening in mammalian cells. Careful consideration of multiplicity of infection (MOI), assay background, and clone penetrance is crucial for successful transdominant selections.

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Setting a Successful Sorting for Extracellular Vesicle Isolation

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Setting a Successful Sorting for Extracellular Vesicle Isolation

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Area of Science:

Mammalian cell genetics
Molecular biology
Biotechnology

Background:

Flow cytometry combined with retroviral expression libraries is a powerful technique for genetic screening in mammalian cells.
This method involves sorting cells based on reporter activity to enrich for specific genetic inhibitors.
It has been successfully applied to isolate peptides and RNAs modulating biochemical pathways.

Purpose of the Study:

To experimentally and theoretically analyze variables affecting enrichment in mammalian cell bioassays using flow cytometry.
To investigate the impact of assay background, fluorescence ratio, genetic penetrance, and multiplicity of infection (MOI) on selection efficiency.

Main Methods:

Utilized a mammalian cell bioassay with a fluorescent reporter system.
Employed retroviral expression libraries for genetic screening.
Conducted experimental analysis and computer modeling to evaluate key variables.
Investigated the role of multiplicity of infection (MOI), assay background, and clone penetrance.

Main Results:

Multiplicity of infection (MOI) measurements were found to be problematic and significantly impact enrichment at later stages.
Assay background and clone penetrance were identified as critical factors throughout the entire enrichment process.
Achieved enrichments approximately twofold of the theoretical maximum.

Conclusions:

MOI determination requires caution due to potential underestimation.
High MOI can be beneficial early in selection but detrimental in later rounds.
MOI, assay background, and clone penetrance are key determinants for successful transdominant selections using fluorescence-activated cell sorting (FACS).