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Flow cytometry combines cell biology with the study of light waves. Indeed, Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology can provide rapid, quantitative, multi-parameter analyses on single living (or dead) cells based on the measurement of visible and fluorescent light emission. Flow cytometry is a widely used method for characterizing properties of biological cells, including size, granularity, cell receptors, and even biological activities, such as DNA, RNA replication, and protein synthesis or suppression.
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This basic protocol focuses on the measurement of fluorescence intensity produced by fluorescent-labeled antibodies and ligands that bind specific cell-associated molecules.
If it is desired to simultaneously analyze surface and intracellular molecules at single-cell level, staining cell-surface antigen first by following procedure A step 1 to 6, then stain the intracellular antigen by following procedure B. Perform all procedure B steps in dark.
This procedure utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells. Propidium Iodide (PI) is a DNA-binding fluorochrome that intercalates in the double-helix. Ribonuclease-A is used to eliminate the staining of double-stranded RNA.
Procedure C can be combined with the procedure A of cell surface antigens analysis. Use only FITC-labeled antibodies for surface antigen, since PI emits in the orange to red region of the spectrum (FITC emits green). Do not fix the cells with paraformaldehyde. Start procedure C after step 6 of procedure A.
Additional FACS Protocol Resources
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