Erythrophagocytosis Assay

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

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 37oC, 6-7% pCO 2 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 37oC, 6-7% pCO 2 , ON (overnigh). Next day wash once and incubate with prepared erythrocytes (RBC) for 1-3 hours in thermostate at 37oC, 6-7% pCO 2. 
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, 10oC, 7 min. 
3. resuspend 2 ml of RBCs in 5 ml of PBS (sterile, ice-cold), place at 4oC, ON. 
4. next day wash 2 times with sterile PBS (centrifugation-resuspension in PBS). 
5. 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. 
6. wash 2 times with PBS (centrifugation-resuspension in PBS). 
7. centrifuge at 1000 rpm, 10oC, 7 min. 
8. take 20 µl 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. NH 4 Cl, 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 NH 4 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%)
   
3. the hypothetical ratio of peritoneal macrophages/peritoneal cells is 1/3. 
4. distinguish phagocytosed RBCs and overposed ones.
   

REFERENCES

Hunter, S., Indik, ZK, Kim, MK, Cauley, MD, Park, JG and Schreiber, AD (1998). Blood 91(5): 1762-8.