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  ] prepare peritoneal macrophages. count peritoneal cells and distribute on 6-wells Grener cell culture plate (2-6x10^6 peritoneal cells/ml), place in thermostate at 37ѓC, 6-7% pCO2 for  3 hours. 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). you may run the erythrophagocytosis right now or, alternatively, change the culture medium and place macrophages in thermostate at 37ѓC, 6-7% pCO2 , ON (overnigh). Next day wash once and incubate with prepared erythrocytes (RBC) for 1-3 hours in thermostate at 37ѓC, 6-7% pCO2. wash 3 more times, add 1 ml of sterile PBS (ice-cold, to stop phagocytosis). 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] collect blood on heparine. centrifuge at 1000 rpm, 10ѓC, 7 min. resuspend 2 ml of RBCs in 5 ml of  PBS (sterile, ice-cold), place at 4ѓC, ON. next day wash 2 times with sterile PBS (centrifugation-resuspension in PBS). resuspend 500 µl of the RBCs in 10 ml of PBS-BSA (1%), add 50 µl of anti-RBC antibody, incubate for 1 hour at RT , apply slight rotation. wash 2 times with PBS (centrifugation-resuspension in PBS). centrifuge at 1000 rpm, 10ѓC, 7 min. take 20 µl of RBC pellet and add to macrophages. SOLUTIONS 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%) PBS PBS-BSA 1% NH4 Cl, 160 mM ADDITIONAL INFO IMDM supplemented with AJ (3-5%), FCS/FBS (5-10%) and heparine (100 U/ml) may be used when purifying macrophages. an additional step of osmatic shock (with 1ml of NH4 Cl, 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%) the hypothetical ratio of peritoneal macrophages/peritoneal cells is 1/3. distinguish phagocytosed RBCs and overposed ones. SCHEME 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.