Erythrophagocytosis

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 37?C, 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 37?C, 6-7% pCO2, ON (overnigh). Next day wash once and incubate with prepared erythrocytes (RBC) for 1-3 hours in thermostate at 37?C, 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, 10?C, 7 min.

3. resuspend 2 ml of RBCs in 5 ml of  PBS (sterile, ice-cold), place at 4?C, 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, 10?C, 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. 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



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.