Flow Cytometry Protocols

Flow Cytometry Workflow: Fluorescence-Activated Cell Sorter (FACS)

Direct Staining Protocol for Flow Cytometry

Note: The following protocol is given to provide a general procedure that can be used as a template to construct a specific protocol for an experimental assay. Investigators are strongly encouraged to collaborate with a flow cytometry facility or technician to fully develop appropriate procedures for their experimental systems.

Items Needed

Cells (~0.5–1 x 106/mL) prepared and counted, in PBS with Ca²⁺ and Mg²⁺ , 1% BSA or 2% FCS.
Directly conjugated monoclonal antibody to desired cell surface marker.
Wash solution (1X Dulbecco's PBS, Ca²⁺ and Mg²⁺ free with and without 1 % BSA)
Optional fixative: 1% formaldehyde-PBS pH 7.4, freshly prepared.

Protocol

Add 50–100 L of cells to each of three 12 x 75 mm polypropylene or polystyrene tubes

Note: The initial amount of cells collected should be adjusted to approximately ~.75 x10⁶ cells per mL so that 50 µ;L added will equal approximately 105 cells per tube.
To the first tube, add the appropriate volume (usually 5–10 µL) of fluorochrome-conjugated monoclonal antibody. To the second tube, add the same volume of matched isotyp control antibody. The isotype control antibody should match the isotype of the conjugated antibody of interest. To the final tube of cells, add the equivalent volume of 1X PBS as conjugated antibody. This will serve as an autofluorescence control to establish the appropriate flow cytometer electronic settings. Vortex each of the sample tubes at moderate speed.
Incubate samples for 20–30 minutes at room temperature or 4°C, in the dark.
Wash cells in tubes with 3 mL volumes of 1X PBS with 1% BSA and centrifuge samples at 400 xg for 10 minutes at room temperature. Carefully pour or pipette off supernatant fluid. This step can be repeated if desired.
Cells can be read without fixing if they are to be used immediately. Simply resuspend the cell pellet in 500 µL of PBS wash solution, and keep at 4°C in the dark until used (usually less than 4 hours). Alternatively, loosen the cell pellet by vortexing it in the residual wash fluid. Add 500 µL of 1% formaldehyde-PBS to each sample and quickly mix again. It is critical to mix the cell pellet prior to adding the fixative; otherwise the cells will become fixed into a solid mass that cannot be sent through the flow cytometer. Fixed samples can be stored at 4°C in the dark until FACS analysis is performed. Fixed samples should be used within 48 hours.

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