Antibody-coated platelets (opsnized) may be used in the subsequent thrombophagocytosis assay.
PROTOCOL
- 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 37ѓC.
- Centrifuge at 3000 rpm at 4ѓC, for 7 min.
- Resuspend in BSGC.
- Centrifuge at 3000 rpm at 4ѓC, for 7 min.
- Resuspend in 200µl of IMDM.
- Centrifuge at 3000 rpm at 4ѓC, for 7 min.
- Resuspend in 200µl of IMDM-FCS 5% decomplemented.
SOLUTIONS
- BSGC
- BSGC-BSA 1% (v/w) = 9990ml BSGC+10g BSA
- IMDM (Iscove's Modified Dulbecco's Medium)
- IMDM-FCS 5% (v/v) heat-decomplemented (56ѓC, 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.
