Intracellular Staining Protocol for Flow Cytometry

Flow cytometry is a tool which is useful in the study of cell proteins. It utilizes the emission of light or fluorescence from the proteins within the cell. Antibodies or probes tagged with fluorescence are used to study specific proteins. Flow cytometry is used to diagnose a number of diseases, including cancer, but is also useful in medical research.

Fixing Cells

• Cells should be fixed to maintain the stability of the intracellular proteins. A number of fixatives can be used such as formaldehyde or ice-cold acetone. Following fixation, the cell membrane needs to be permealized to allow the antibody to enter the cell. Detergents such as Triton or NP-40, or ice-cold methanol can be used to make holes in the cell membrane.

Staining

• Wash cells in the blocking buffer 0.5 percent bovine serum albumin diluted in phosphate buffered saline, BSA/PBS. Cells are incubated in blocking buffer to reduce nonspecific binding. Add primary antibody diluted in blocking buffer and leave to incubate at room temperature, for optimized length of time. Wash with blocking buffer.

Analysis

• Incubate the secondary antibody, which is labeled with a fluorescent tag, at room temperature for 30 minutes. To remove unbound antibody wash cells in blocking buffer. Spin cells into a pellet and then resuspend in PBS and analyze on flow cytometer.