Ascitic fluid (also called ascites) is an intraperitoneal fluid
extracted from mice that have developed a peritoneal tumor. For
antibody production, the tumor is induced by injecting hybridoma cells
into the peritoneum, which serves as a growth chamber for the cells. The
hybridoma cells grow to high densities and continue to secrete the
antibody of interest, thus creating a high-titered solution of
antibodies for collection. Antibody concentrations will typically be
between 1 and 10mg/ml.
- Prime adult female mice (at least 6 weeks old) of the same genetic background as your hybridomas by injecting 0.5mL of pristane (2,6,10,14-tetramethyldecanoic acid) into the peritoneum. These solutions will act as irritants to the mice, which respond by secreting nutrients and recruiting monocytes and lymphoid cells into the area. This creates a good environment for the growth of the hybridoma cells.
- After 7-14 days, inject 5x10(5) to 5x10(6) hybridoma cells ip. Prior to injection, the cells should be growing rapidly. Centrifuge the cells and wash once in PBS. Inject the cells in no more than 0.5mL of PBS.
- Ascitic fluid may begin to build up within 1-2 weeks following the injection of the cells. Tap the fluid when the mouse is noticeably large, but before the mouse has difficulty moving. Carefully withdraw as much fluid as possible with 18-gauge needle attached to a 5mL syringe.
- Return the mouse to its cage. Many mice will produce a second or third batch of ascitic fluid. You can also bleed out the mouse and combine the blood with the ascitic fluid.
- Incubate the fluid at 37°C for 1 hour. Transfer to 4°C overnight.
- Spin the fluid at 3000g for 10min. If there is an oil layer, remove this first and discard. Carefully remove the supernatant from the cell pellet. Spin again if necessary.