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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

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OUTLINE

The erythrophagocytosis assay is performed to compare the phagocytic rate with and without anti-eythrocyte antibody in either macrophages from control animals or virus-infected counterparts.

PROTOCOL

[  erythrophagocytosis  ]
  1. prepare peritoneal macrophages.
  2. count peritoneal cells and distribute on 6-wells Grener cell culture plate (2-6x10^6 peritoneal cells/ml), place in thermostate at 37C, 6-7% pCO2 for  3 hours.
  3. aspirate the supernatant (SN), add 2 ml of  IMDM-AJ (3-5%)-FCS/FBS (5-10%) and aspirate the SN, repeat 1 more time (washing procedure).
  4. you may run the erythrophagocytosis right now or, alternatively, change the culture medium and place macrophages in thermostate at 37C, 6-7% pCO2, ON (overnigh). Next day wash once and incubate with prepared erythrocytes (RBC) for 1-3 hours in thermostate at 37C, 6-7% pCO2.
  5. wash 3 more times, add 1 ml of sterile PBS (ice-cold, to stop phagocytosis).
  6. count macrophage as positive if 5 and more RBCs have been phagocytosed (on invert microscope). Estimate the rate of phagocytosis as a proportion of positive cells to overall calculated cells, %.
[preparation of erythrocytes]
  1. collect blood on heparine.
  2. centrifuge at 1000 rpm, 10C, 7 min.
  3. resuspend 2 ml of RBCs in 5 ml of  PBS (sterile, ice-cold), place at 4C, ON.
  4. next day wash 2 times with sterile PBS (centrifugation-resuspension in PBS).
  5. resuspend 500 l of the RBCs in 10 ml of PBS-BSA (1%), add 50 l of anti-RBC antibody, incubate for 1 hour at RT , apply slight rotation.
  6. wash 2 times with PBS (centrifugation-resuspension in PBS).
  7. centrifuge at 1000 rpm, 10C, 7 min.
  8. take 20 l of RBC pellet and add to macrophages.
SOLUTIONS
  1. IMDM [(Iscove's Modified Dulbecco's Medium with 25 mM Hepes/with L-Glutamine) BioWhittaker Europe Cat.: BE12-722F]-AJ(3-5%)-FCS/FBS (5-10%)
  2. PBS
  3. PBS-BSA 1%
  4. NH4Cl, 160 mM
ADDITIONAL INFO
  1. IMDM supplemented with AJ (3-5%), FCS/FBS (5-10%) and heparine (100 U/ml) may be used when purifying macrophages.
  2. an additional step of osmatic shock (with 1ml of NH4Cl, for less than 3 min) may be added right after the first centrifugation at the step of macrophage preparation with a consequent neutralization step with IMDM-AJ (3-5%)-FCS/FBS (5-10%)
  3. the hypothetical ratio of peritoneal macrophages/peritoneal cells is 1/3.
  4. distinguish phagocytosed RBCs and overposed ones.
SCHEME
erythrophagocytosis
enlarge  ]

REFERENCES

Hunter, S., Indik, Z.K., Kim, M.K., Cauley, M.D., Park, J.G. and Schreiber, A.D. (1998). Inhibition of Fcgamma receptor-mediated phagocytosis by a nonphagocytic Fcgamma receptor. Blood 91(5): 1762-8.


created by Andrei Musaji          ::          updated : 2004