Cell Surface Staining Protocol
Cell Harvesting
Spin down cell suspension at 1000 RPM for 5 minutes and decant supernatant. Resuspend thepellet in 1X PBS. Count the cells with a hemocytometer. Add the total desired number of cells
to a flow tube (generally 0.5-1 million cells per sample). Wash the cells by adding ~1 ml (ormore if many samples) of 1X PBS to the flow tube. Spin down cell suspension at 1000 RPM for
5 minutes and decant supernatant. Gently tap the tube to loosen the cell pellet. Add 50 ul
staining buffer per 1 million cells. Add 50 ul of cell suspension to the desired number of flow
tubes.

Staining-Primary
Add the full amount of antibody to 50 ul of staining buffer and add this to the 50 ul of cellsuspension, pipetting up and down to mix.
a. Fluorescent labeled primary antibody: Incubate on ice for 30-60 min; protect from light during
incubation.
b. Unlabeled primary antibody: Incubate on ice for 30-60 min.

-Secondary (unlabeled primary only)
Add ~1 ml of staining buffer and spin down cells at 1000 RPM for 5 min. Decant supernatant
and wash cells twice with 1-2 mls of staining buffer. Resuspend each cell pellet with 100 ul of
secondary antibody solution. Incubate on ice for 30 min; protect from light during incubation.

Final Wash and Data Acquisition
Wash cells twice with 1-2 ml of staining buffer. After the final decanting, resuspend stained cells
in an appropriate amount of staining buffer. Acquire data on a flow cytometer following
manufacturer’s recommendations.

Intracellular Staining Protocol
Cell Harvesting
Spin down cell suspension at 1000 RPM for 5 minutes and decant supernatant. Resuspend thepellet in 1X PBS. Count the cells with a hemocytometer. Add the total desired number of cells
to a flow tube (generally 0.5-1 million cells per sample). Wash the cells by adding ~1 ml (ormore if many samples) of 1X PBS to the flow tube. Spin down cell suspension at 1000 RPM for
5 minutes and decant supernatant. Gently tap the tube to loosen the cell pellet.

FLOW CYTOMETRY PROTOCOL | Copyright NSJ Bioreagents

Fixation and Permeabilization
Resuspend the cell pellet with 50 ul of 1X fixation buffer per 1 million cells. Incubate at room
temperature in the dark for 30 min, then spin down cells at 1000 RPM for 5 min and decant
supernatant. Resuspend the cell pellet with 1-2 mls of 1X permeabilization buffer and spin downcells at 1000 RPM for 5 min. Decant supernatant and repeat wash step. Dump off supernatantand add 50 ul of 1X permeabilization per 1 million cells in the pellet. Pipette up and down toevenly suspend cells. Add 50 ul of cell suspension to the desired number of flow tubes.

Staining-Primary
Add the full amount of antibody to 50 ul of permeabilization buffer and add this to the 50 ul ofcell suspension, pipetting up and down to mix.
a. Fluorescent labeled primary antibody: Incubate at room temperature for 30-60 min; protectfrom light during incubation.
b. Unlabeled primary antibody: Incubate at room temperature for 30-60 min.

-Secondary (unlabeled primary only)
Add ~1 ml of permeabilization buffer and spin down cells at 1000 RPM for 5 min. Decant
supernatant and wash cells twice with 1-2 mls of permeabilization buffer. Resuspend each cellpellet with 100 ul of secondary antibody in permeabilization buffer. Incubate at room
temperature for 30 min; protect from light during incubation.

Final Wash and Data Acquisition
Wash cells twice with 1-2 ml of staining buffer. After the final decanting, resuspend stained cells
in an appropriate amount of staining buffer. Acquire data on a flow cytometer following
manufacturer’s recommendations.

FLOW CYTOMETRY PROTOCOL | Copyright NSJ Bioreagents