Antibody Titration

Harvest cells and prep as usual.

Add 1e6 cells to the wells of a round bottom 96 well plate for each antibody dilution series being tested in addition to an extra well for an unstained negative control.

Centrifuge plate 3” @ 1500 rpm (this speed is for splenocytes. You may need to adjust this for your particular cell type), flick and vortex.

Add 50 uL FC blocker to each well and place plate on ice for 10”. (If your antibody is an Fab fragment this step would not be necessary)

Prepare serial doubling dilutions by adding 98 uL of staining wash buffer (SWB) to the first tube of each series and 50 uL SWB to the remaining tubes.

Add 2uL of stock antibody solution to the first tube for an initial 50:1 dilution.

Perform serial doubling dilutions for each antibody by removing 50 uL from the first tube and placing it in the second tube, and so on for a series of dilutions from 50:1 down to 6,400:1 if using all 8 wells in a column of a 96 well plate.

Centrifuge the FC blocked cells, flick and vortex.

Add 50 uL of each antibody dilution to the appropriate wells containing the cells. (add 50 uL of SWB without any antibody to the negative, unstained control well)

Incubate plate on ice for 20”, keeping covered. (either put a lid on the ice bucket or cover the plate with foil as the fluorochromes are light sensitive)

Centrifuge and wash plate 2X with 150-200 uL SWB.

If the antibodies are biotinylated add 50 uL Streptavidin-FITC, PE, etc. and incubate another 20 to 30 minutes on ice. ( If not using biotinylated antibodies proceed to the last step.)

Wash 2X as in step nine.

Resuspend cells in 90 uL of SWB and then add 10uL 10% paraformaldehyde and keep on ice and in the dark until FACS analysis and titration results interpretation.

Staining Wash Buffer

60 mls 10X PBS

6 ml 10% Sodium Azide

12 ml FCS

522 ml dd H2O