Surface Staining Protocol

1. Take 1 x 10⁶ cell suspension, spin down, and discard supernatant.
2. Add 1mg of Fc block /10⁶ cells and incubate on ice for 5 min.
3. Add surface staining antibodies or isotype controls of choice and incubate 30 min. on ice.
4. Wash cells with 2 ml of PBS/2%FCS, spin down, and discard supernatant.
5. Slowly vortex pellet and add 1 ml of 1% paraformaldehyde.
6. Wrap samples in foil and store at 4°C.

Titration of Abs and blocking reagents should be performed.

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Combination of Surface & Intracellular (Indirect) Staining

1. Take 1 x 10⁶  cell suspension, spin down, and discard supernatant.
2. Add 1mg of Fc block /10⁶ cells + *normal serum  incubate on ice for 10 min. Spin down 5 min. at 1200 RPM and discard all liquid.
3. Do surface staining at this point, add surface staining antibody of choice, then wash cells 1X with PBS/2%FCS, and discard all liquid.
4. VORTEX before adding Cytofix.
5. Resuspend cells in 100 ul of Cytofix/Cytoperm solution, VORTEX, and incubate on ice for 20 min.
6. Wash 2X with 1ml BD Perm/wash buffer (1:10 dilution in water) (1ml/1 x 10⁶)
7. Add primary antibody of choice and incubate on ice for 30 min.
8. Wash 1X with 1 ml BD perm/wash
9. Add Fc block + *normal serum.
10. Add secondary antibody of choice and incubate on ice for 30 min.
11. Wash 1X with 1ml of BD perm/wash buffer. Spin down and discard liquid.
12. Wash cells in 1 ml PBS/2%FCS, spin down, and resuspend in 1 ml PBS/2%FCS.
13. Wrap and store at 4°C. Analyze as soon as possible (<72hrs)

(*)Blocking serum must be from species where the secondary Ab was originated. Ex: if your 2º Ab is goat anti-mouse IgG, you should use normal goat serum.

Titration of Abs and blocking reagent should be performed.