Production of Ascitic Fluids in Mice

1. Prime BALB/c mice with 0.5 mL of Pristane injected i.p. prior to hybridoma cell injection.

2. 7 d after Pristane administration, inject i.p. 5 X 106 to 5 X 107 prewashed (twice in serum-free DMEM) hybridoma cells.

3. Check inoculated mice for abdominal distension every 3-5 d until the abdomen is filled completely with ascitic fluid (typically, 3-4 wk). Drain ascitic fluid from the abdomen using an 18-gage needle.

4. Clear ascitic fluids by centrifugation at 500g for 10 min; remove the yellowish layer of fat on the top of the ascitic fluid and clotted material from other areas of the tube.

5. Aspirate the supernatant, aliquot, and keep at -20°C until further analysis and MAb purification.

6. Collect the cell pellet and use it one or two times to induce ascites in the next group of Pristane-primed animals. Do not use ascitic cells more than twice.

7. In most labs, one mouse produces 5-10 mL of ascitic fluid containing 5-20 mg/mL of the MAb.

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