OUTLINE
This modification of peritoneal macrophage preparation is designed for a subsequent thrombophagocytosis test.
PROTOCOL
- Sacrifice a mouse (use dry ice, CO2)
- Sink a mouse in the 5% water solution of Dettol (Chloroxylenol) for 1 min, at RT.
- Dry the fur thoroughly. Inject i.p. 5 ml of ice-cold IDDM-FCS (5%, decomplimented). Gently massage the anterior and lateral walls of the abdomen.
- Dissect the tissues and open the peritoneum cavity. Accurately collect the peritoneum washes on to the Petri dish placed on ice.
- Filter the washes thorough a nylon filter into the 15 ml Falcon tube placed on ice.
- Centrifuge at 1200 rpm, at 4ѓC, for 5 min.
- Resuspend in IMDM-FCS (5%, decomplimented)
- Count cells (do not count erythrocytes). Adjust the concentration of counted cells as 20x10^6/ml
SOLUTIONS
- IMDM-FCS 5%, decomplemented (heat for 30 min at 56ѓC)
ADDITIONAL INFO
- All solutions should be sterile!
- Add NH4Cl, 160 mM right after the first centrifugation if the osmatic shock needed (elimination of erythrocytes).
SCHEME
REFERENCES
- Celada, A. and Nathan, C. (1994). Macrophage activation revisited. Immunol Today 15(3): 100-2.
- Gordon, S., Keshav, S. and Chung, L.P. (1988). Mononuclear phagocytes: tissue distribution and functional heterogeneity. Curr Opin Immunol 1(1): 26-35.
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