Antibodies and flow cytometry

Cell surface FACS staining protocol

1. Resuspend 1x10⁶ PBMCs in 100 μl cold PBS.
2. Add 2-5ul fluorophore-conjugated antibodies of cell-surface proteins.
3. Incubate for 30 min at 4°C in the dark.
4. Wash the cells twice with 1 ml PBS by centrifugation at 400xg for 5 minutes and resuspend them in 200 μl 2% PFA. Keep the cells in the dark on ice or at 4°C in a fridge until your scheduled time for analysis.

Intracellular staining protocol

1. First perform cell surface staining as described above.
2. Fix cells in 0.25 ml/tube Fixation/Permeabilization solution (BD Biosciences) for 20 minutes at 4°C in the dark.
3. Wash cells twice with 1 ml 1×Perm/Wash buffer (BD Biosciences) by centrifugation at 400xg for 5 minutes.
4. Resuspend fixed/permeabilized cells in 100 μl 1×Perm/Wash buffer.
5. Add 1-2 μl fluorophore-conjugated antibodies of intracellular proteins and incubate for 30 minutes at 4°C in the dark.
6. Wash twice with 1 ml of 1×Perm/Wash buffer by centrifugation at 400xg for 5 minutes.
7. Resuspend fixed and intracellularly labeled cells in 200 μl 2% PFA and keep them in the dark at 4°C in a fridge until analysis.

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