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Site Density Protocol   

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Abstract

The site densities of cell surface molecules are useful information for cell function analysis. Using antibody staining and commercial available calibration beads, this assay quantitatively determines the T cell receptor site density at the single T cell level. This method can be easily extended to quantify other surface molecule densities on different cells or beads.

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.  
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Huang, J. (2012). Site Density Protocol. Bio-101: e234. DOI: 10.21769/BioProtoc.234.
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