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Flow Cytometry Protocol for Indirect Intracellular Staining of Cultured Cells Grown in Suspension.
16% paraformaldehyde (PFA) (Electron Microsopy Sciences RT 15710)
90% Methanol (ice cold)
PBS (Phosphate Buffered Saline pH 7.2)
ICSB (IntraCellular Staining Buffer) – 1% FBS, 0.05% Sodium Azide in PBS
Cell Fixation and Permeabilization:
For cells grown in suspension, add fresh 16% PFA to achieve a final concentration of 1.54% directly to the cells in media (1-2 x 10
Fix cells at room temperature (RT) for 10 minutes.
Incubate and fix on ice for an additional 30 minutes.
Centrifuge fixed cells at 250 x g for 5 minutes at 20
Pour off media + PFA into dedicated PFA waste containier.
Permeabilize cells by resuspending in ice cold 90% methanol to a final cell concentration of 1 X 10
Cells may be stored at -70
C for up to a month.
Aliquot 1 ml (1 x 10
cells) of cells in methanol for each tube/sample.
Centrifuge at 250 x g for 5 minutes at room temperature and remove supernatant (perform all subsequent spins at these conditions)
Resuspend cells in 1 ml ICSB
Centrifuge cells and remove supernatant
Resuspend the cell pellet in 100µl of ICSB containing diluted primary antibody per Bethyl’s recommendation
Incubate on ice for 1 hour
Rinse the cells 2X in ICSB by centrifugation
Resuspend in 100µL of conjugated secondary antibody per Bethyl’s recommendation
Incubate on ice for 30 minutes in the dark
Rinse the cells 2X in ICSB by centrifugation. Continue to minimize exposure of cells to the light
Resuspend in 300µl ICSB in the dark
Filter cells through a strainer to remove clumps
Analyze with a flow cytometer