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.

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.