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viral immunity and pathogenesis group. macrophage preparation protocol


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  ::  p e r i t o n e a l  m a c r o p h a g e  p r e p a r a t i o n  ::


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OUTLINE

This modification of peritoneal macrophage preparation is designed for a subsequent thrombophagocytosis test.

PROTOCOL

  1. Sacrifice a mouse (use dry ice, CO2)
  2.  Sink a mouse in the 5% water solution of Dettol (Chloroxylenol) for 1 min, at RT.
  3. 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.
  4. Dissect the tissues and open the peritoneum cavity. Accurately collect the peritoneum washes on to the Petri dish placed on ice.
  5. Filter the washes thorough a nylon filter into the 15 ml Falcon tube placed on ice.
  6. Centrifuge at 1200 rpm, at 4ƒC, for 5 min.
  7. Resuspend in IMDM-FCS (5%, decomplimented)
  8. Count cells (do not count erythrocytes). Adjust the concentration of counted cells as 20x10^6/ml
SOLUTIONS
  1. IMDM-FCS 5%, decomplemented (heat for 30 min at 56ƒC)

ADDITIONAL INFO

  1. All solutions should be sterile!
  2. Add NH4Cl, 160 mM right after the first centrifugation if the osmatic shock needed (elimination of erythrocytes).

SCHEME
macrophage preparation
[  enlarge  ]

REFERENCES

  1. Celada, A. and Nathan, C. (1994). Macrophage activation revisited. Immunol Today 15(3): 100-2.
  2. Gordon, S., Keshav, S. and Chung, L.P. (1988). Mononuclear phagocytes: tissue distribution and functional heterogeneity. Curr Opin Immunol 1(1): 26-35.
created by Andrei Musaji          ::          updated : 2004