Site Density Protocol   

Abstract

Materials and Reagents

1. PE-conjugated anti-mouse TCR Va2 monoclonal antibody B20.1 (BD)
2. PE Rat IgG2a, λ Isotype Control (BD)
3. QuantiBRITE PE beads tube (BD)

Equipment

1. Countertop Centrifuge
2. BD LSR flow

Procedure

1. OTI T cells (1x105~1x106) were incubated with anti-TCR Va2 antibody or isotype control antibody at 10 μg/ml (or saturated concentration) in 200 μl of FACS buffer at 4 oC for 30 min on a shaker;
2. Wash three times with cold FACS buffer by centrifuge at 500g for 3 minutes;
3. Resuspend the T cells in 400 μl cold FACS buffer;
4. Add 400 μl cold FACS buffer into QuantiBRITE PE tube, and gently shake the tube to resuspend the beads;
5.  Measure the fluorescence intensities of T cells and QuantiBRITE PE beads by a BD LSR flow cytometer;
6. Plot a linear regression of PE molecules per bead against measured mean fluorescence, using the following equation:
   y = mx + c
   Where y equals measured mean fluorescence and x equals PE molecules per bead provided by manufacturer; m is slope and c is the intercept.
7. Use above equation to calculate the total number of molecules per cell according to measured T cell mean fluorescence (after subtract isotype control fluorescence) and the antibody F/P (the number of fluorochromemolecules per Ig molecule) molar ratio, and divided by the T cell surface area to obtain the site density.

Recipes

1. FACS staining Buffer: 
   PBS
   5 mM EDTA
   1% BSA
   0.02% sodium azide
   

References

1. Huang J., Zarnitsyna V.I., Liu B., Edwards L.J., Jiang N., Evavold B.D., Zhu C. (2010). The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness. Nature 464(7290): 932-6.