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%
- NH4Cl, 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 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%)
- the hypothetical ratio of peritoneal macrophages/peritoneal cells is 1/3.
- distinguish phagocytosed RBCs and overposed ones.
SCHEME REFERENCESHunter, S., Indik, Z.K., Kim, M.K., Cauley, M.D., Park, J.G. and Schreiber, A.D. (1998). Blood 91(5): 1762-8.
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