Cell surface staining protocol

FACS buffer (FB) recipe:

1x HBSS with 2% FBS and 12 mM HEPES. Optional 0.1% sodium azide to prevent microbial growth and 1 mM EDTA to prevent clumping.

Protocol:

1. Harvest, count and wash your filtered single cell suspension.
2. Aliquot 0.1–0.5 x 10⁶ cells into conical polystyrene 5 mL tubes or 96-well round or V-bottom plates for staining.
3. Add FB and spin at 1100 rpm for five minutes.
4. Prepare appropriate dilutions of antibodies in FB and add 50-100 uL per sample.
5. Stain in the dark on ice or at 4°C for 30 minutes.
6. Wash twice with FB.
7. Resuspend in 500 uL FB with a live dead marker such as PI, DAPI or 7-AAD.
8. In order to prevent clogging of the flow cytometer, re-filter the cell suspension through a 70 μm cell strainer if clumps can still be observed.

Notes:

1. Cells expressing high levels of Fc receptors will contribute to non-specific binding. Fc blocking is recommended after step 3 and prior to staining with specific antibodies. Fc block should be used according to the manufacturer’s directions.
2. Appropriate controls need to be prepared and run prior to acquiring your fully stained samples. For multi-colour experiments, single stained controls for each fluorophore are mandatory in order to set up compensation.
3. In order to accurately gate cells when using a multi-colour panel, fluorescence minus one (FMO) controls are ideal. Fluorescence minus one controls allows you to identify appropriate gating strategies in the context of data spread due to numerous fluorochomes. An FMO contains all the fluorophores except the one of interest. For example, FITC FMO control contains all fluorophores in the stain except FITC.