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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 37ƒC.
  2. Centrifuge at 3000 rpm at 4ƒC, for 7 min.
  3. Resuspend in BSGC.
  4. Centrifuge at 3000 rpm at 4ƒC, for 7 min.
  5. Resuspend in 200µl of IMDM.
  6. Centrifuge at 3000 rpm at 4ƒC, 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 (56ƒC, 30min).

SCHEME
plab
[  enlarge  ]

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.
created by Andrei Musaji          ::          updated : 2004