Antagene inc. offers ascites production from small Research and Development scales to large-scale production. We secured a USDA-registered research facility, and we comply with all federal guidelines for the care and use of laboratory animals.

Priming and Cell Preparation:
You may provide sufficient cells from the designated cell line (approximately 2–2.5 x 106 cells per animal). A minimum of five mice will be used per hybridoma clone. As soon as we receive your order, we will start to prime at least 10 mice per hybridoma clone with Pristane for you. (Default: minimum 14 days of priming with 0.5 ml of Pristane per mouse.) The time interval between priming and inoculation of hybridoma cells is 10–14 days. Generally, very high concentrations of cells are associated with greater mortality, and concentrations below 1 x 105 cells elicit fewer ascitic tumors, and these tend to have a smaller volume yield. Cell suspensions should be prepared sterilely in physiological solutions.

If you prefer to send us frozen vials, 3 vials of cells (3x106 cells per vial) from the designated cell line are required. As soon as we received the sample, Cells were thawed and viability tested. We then begin to expand cells to the appropriate number for inoculation (Default: 2-3 X 106 cells per animal). We determine percent viability via Trypan Blue Dye. Approximately one or two weeks are needed to prepare cells from frozen stocks for injection, and there is an additional charge of $300 per clone for this service.
(Additional: Nude and SCID mice are available for ascites production for non-murine hybridoma cell lines.) 

Harvesting Ascites Fluid:
Hybridomas are injected into pristane-primed mice, and then mice are monitored daily for ascites development and general condition. Ascites collection begins when the accumulation of ascites becomes evident and continues until the final harvests are performed. You can choose to receive unpurified, defibrinated ascites, or Antagene will purify ascites for you. In general, ascites production is completed within 2–3 weeks after the injection of hybridomas.

1. Cells are harvested in the logarithmic phase of growth and injected into mice.
2. Approximately 2 weeks after cell inoculation, tapping will begin. Mice will be held for up to 6 weeks after cell injection.
3. Mice receive a maximum of three needle taps with a terminal tap.
4. Daily taps are pooled and then centrifuged at low speed.
5. Daily taps are stored frozen at -70° C in 50-ml centrifuge tubes.
6. At the completion of tapping, centrifuge tubes from daily taps are thawed and pooled.
7. The final pooled material receives high-speed centrifugation.

Deliverable:
The ascites fluid will be sent back to you via overnight transporter. If you ask for antibody purification, we will use those ascites to purify antibodies (additional charge of $500 for IgG purification), and 15-20 mg of purified antibodies will be delivered at the end. Shipment costs to your laboratory are additional, as indicated in our invoice.

Confidentiality:
We will keep no sample after the experiment's achievement. No sample will be transferred to any third party or be commercially used. You will remain the only owner of the hybridoma. Moreover, we will not disclose to any third party that we have used your material for any manipulation or worked with your company. Starting with your own hybridoma, we cannot guarantee ascites quality in cases of low antibody yield.

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