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viral immunity and pathogenesis group. thrombophagocytosis.


l a b o r a t o r y   r e s e a r c h   i n   i m m u n o l o g y   ::   v i r o l o g y   ::   h e m a t o l o g y
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v i r a l   i m m u n i t y   &   p a t h o g e n e s i s   g r o u p

::  p r o t o c o l s 
::  t h r o m b o p h a g o c y t o s i s  ::







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.

  1. Add 100l of peritoneal cells (20x10^6/ml) into 5 ml Polypropylene Round-Bottom tubes (Falcon, Cat.: 35 2063). Add 45 l of platelet suspension resulted from CMFDA-labeling and Ab-coating step. Vortex at medium speed.
  2. Incubate tubes as duplicates at 37C and on ice for 60 min.
  3. To stop the phagocytosis the tubes incubated at 37C should be placed on ice all together at the same time (!).
  4. Add 100l of ice-cold quenching solution to the each tube. Vortex at the medium speed.
  5. Add 4 ml of ice-cold PBS or washing solution.
  6. Centrifuge at 1200 rpm, at 4C, for 5 min.
  7. Resuspend in 4 ml of ice-cold PBS or washing solution.
  8. Centrifuge at 1200 rpm, at 4C, for 5 min.
  9. Resuspend in 4 ml of RT lysing solution. Incubate at RT, for 10 min., at dark.
  10. Centrifuge at 1200 rpm, at 4C, for 5 min.
  11.  Resuspend in 4 ml of ice-cold PBS or washing solution.
  12. Centrifuge at 1200 rpm, at 4C, for 5 min.
  13. Resuspend in 200l of ice-cold DNA-staining solution. Vortex gently.
  14. Run FACS within the next 60 min.
SOLUTIONS
  1. PBS
  2. washing solution (commercial, comes with the kit)
  3. lysing solution (commercial, comes with the kit)
  4. 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
thrombophagocytosis
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