Fluorescent proteins to determine gene expression and transfection efficiency in both live and fixed cells are widely used in flow cytometry, and are particularly useful when performing cell sorting experiments. They can be used as reporters of transcription factors, promoter activity and cellular expression patterns as well as screening for RNAi and CRISPR activity due to the high throughput capacity of flow cytometry. Fluorescent proteins can also be photactivatable, photoswitchable and used in FRET experiments. Initially only green fluorescent protein, there are now over a hundred fluorescent proteins which excite and emit at various wavelengths, making them perfect for multicolor flow cytometry.
Although flow cytometry can quantify expression of markers both on and in cells they do not provide information on the cell concentration or necessarily absolute quantification. To overcome this, fluorescent beads can be added and counted. If a known amount of beads at a known concentration are added to your sample and acquired, the number of beads will be relative to the number of cells. Some cytometers can give accurate cell counts by measuring the volume of sample acquired and in this case the number of cells per ml can be measured.
Internalization of particles, cell surface markers and antigens can occur through various cellular processes, e.g. phagocytosis. Flow cytometry has proved to be an effective method of quantifying this through fluorescently labeling the particle that is to be internalized. Utilizing dyes which either alter their fluorescent characteristics when internalized, or by quenching surface bound fluorescence, the difference between surface and internalized particles can be measured.
Fluorescence in situ hybridization (FISH) was first performed in flow cytometry in the late 1990’s to determine telomere length. Fluorescent nucleic acid probes were used to highlight specific repeat sequences and then fluorescence measured using specific software. Since then RNA expression protocols have been developed which allow quantification of the levels of mRNA. This is a powerful tool as it can be performed in combination with surface staining to identify specific cells and subsets, whereas quantitative RTPCR, whilst very sensitive, will only give information on a cell population.
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