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OUTLINE
The protocol shown below represents an Ab-mediated thrombophagocytosis modification.
PROTOCOL
I. Collect platelets.
II. Label platelets with CMFDA.
III. Coat platelets with an Ab directed to platelet Ag.
IV. Prepare peritoneal macrophages.
V. Run Ab-mediated thrombophagocytosis.
- Add 100µl of peritoneal cells (20x10^6/ml) into 5 ml Polypropylene Round-Bottom tubes (Falcon, Cat.: 35 2063). Add 45 µl of platelet suspension resulted from CMFDA-labeling and Ab-coating step. Vortex at medium speed.
- Incubate tubes as duplicates at 37ƒC and on ice for 60 min.
- To stop the phagocytosis the tubes incubated at 37ƒC should be placed on ice all together at the same time (!).
- Add 100µl of ice-cold quenching solution to the each tube. Vortex at the medium speed.
- Add 4 ml of ice-cold PBS or washing solution.
- Centrifuge at 1200 rpm, at 4ƒC, for 5 min.
- Resuspend in 4 ml of ice-cold PBS or washing solution.
- Centrifuge at 1200 rpm, at 4ƒC, for 5 min.
- Resuspend in 4 ml of RT lysing solution. Incubate at RT, for 10 min., at dark.
- Centrifuge at 1200 rpm, at 4ƒC, for 5 min.
- Resuspend in 4 ml of ice-cold PBS or washing solution.
- Centrifuge at 1200 rpm, at 4ƒC, for 5 min.
- Resuspend in 200µl of ice-cold DNA-staining solution. Vortex gently.
- Run FACS within the next 60 min.
SOLUTIONS
- PBS
- washing solution (commercial, comes with the kit)
- lysing solution (commercial, comes with the kit)
- DNA-staining solution (commercial, comes with the kit)
ADDITIONAL INFO
1. Use PHAGOTEST Kit without E.coli for this assay (Orpegen GmBH, Germany). [ read more ]
2. It's of great importance to follow the temperature recommendations.
SCHEME
[ 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.
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