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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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