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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.

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[Bio101] Site Density Protocol

Immunology > Immune cell function > Lymphocyte

[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)

 

Equipments

 

1.         Countertop Centrifuge

2.         BD LSR flow cytometer

 

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.

 

 



How to cite this protocol: Huang, J. (2012). Site Density Protocol. Bio-protocol Bio101: e234. DOI: 10.21769/BioProtoc.234; Full Text



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