CoatingofPlateletswithAntibodyinvitro

OUTLINE


Antibody-coated platelets (opsnized) may be used in the subsequent thrombophagocytosis assay. 

PROTOCOL

1. Resuspend 1.6x10^8 of CMFDA-labeled platelets in 200μl of BSGC-BSA (v/w 1%) either with a control Ab or an Ab directed to platelet Ag (you may use Grener F-bottom microtitration plate for this step). If the Ab used is rat IgG1 (k) against mouse CD41 (MWReg30, Becton Dickinson, Cat.: 09911D) then the advised concentration is 0.6μg/ml (0.15μg per 200μl of suspension). Incubate the suspension for 45 min., at 37?C.

2. Centrifuge at 3000 rpm at 4?C, for 7 min.

3. Resuspend in BSGC.

4. Centrifuge at 3000 rpm at 4?C, for 7 min.

5. Resuspend in 200μl of IMDM.

6. Centrifuge at 3000 rpm at 4?C, for 7 min.

7. Resuspend in 200μl of IMDM-FCS 5% decomplemented.
   

SOLUTIONS

1. BSGC
   

2. BSGC-BSA 1% (v/w) = 9990ml BSGC+10g BSA

3. IMDM (Iscove's Modified Dulbecco's Medium)
   

4. IMDM-FCS 5% (v/v) heat-decomplemented (56?C, 30min).
   

SCHEME



REFERENCES

Wiener, E., Abeyakoon, O., Benchetrit, G., Lyall, M., Keler, T. and Rodeck, C.H. (2003). Anti-HPA-1a-mediated platelet phagocytosis by monocytes in vitro and its inhibition by Fc gamma receptor (FcgammaR) reactive reagents. Eur J Haematol 70(2): 67-74.