Order 6 six week old Balb/C mice and let the ARC know they are coming. Have your antigen ready for when they arrive. Once they get there earmark the mice and perform a pre-bleed on them to be used as an ELISA control for monitoring the titer and screening of the hybridomas:

Bleeding Mice

1. Place the mouse in a mouse restrainer.
2. Sterilize the tail with 70% ethanol.
3. With a razor blade, nip off the last 2 mm of the tip of the tail.
4. Using a milking motion, pull blood down and let drip off the end of the tail until you have collected ~200 µL. (You may have to pre-bleed twice, with a week or so between bleeds).
5. Take the collected blood and place at 37 °C for 30 min. to remove complement.
6. Place blood at 4 °C overnight to clot.
7. Centrifuge samples at 10,000g 10 min.
8. Pipet off the serum supernatant. Store at -20 °C. This is your pre-bleed control.

Hybridoma Fusion

For one fusion you will need:

1. 8 500 mL bottles of DMEM media
2. 3 500 mL bottles of fetal calf serum (FCS)
3. 2 50 mL bottles of 100X penicillin-streptomycin
4. 10 mL of 100X HAT selection solution
5. 10 mL of 100X HT solution
6. 10 sterile flat-bottomed 96-well plates

Assaying for Positive Clones

Run an ELISA.html">indirect ELISA using the antigen you want the MAb directed against. If you are raising the MAbs against a small hapten that you coupled to a carrier protein for immunization of the mice, then use the hapten coupled to a different carrier protein for the screen.

Expanding Positive Clones

1. The day prior to any expansion, obtain feeder cells. For this first expansion from the 200 µL cultures in 96-well plates to 500 µL cultures in 24-well plates, add 200 µL of feeder cells/well in DMEM/20% FCS HT to the 24-well plates the day before expansion. Let the feeder cells grow overnight in the CO2 incubator.
2. For the initial expansion, resuspend the positive testing hybridoma cells and place all but the last 10 µL (200 µL) in the well of the 24-well plate with the feeder cells. Then bring to 500 µL with DMEM/20% FCS HT.
3. Add 200 µL of DMEM/20% FCS HT to the little bit of cells left in the 96-well plate. This serves as a backup for your positive clones.
4. Place all plates in the CO2 incubator.
5. When the cells are beginning to acidify the media (gets orange-yellow), change the media and double the culture volume by pipetting off 400 µL, and replace with 900 µL fresh DMEM/20% FCS HT.
6. Harvest more feeder cells for your next expansion. Put 0.5 mL f feeder cells in DMEM/20% FCS (note the removal of the HT) in a T25 flask (one for each positive clone) for later use
7. When the cells are ready for expansion out of the 24-well plate, resuspend them and pipet to the T25 flasks with feeder cells. Again leave a little bit in the 24-well plate and refill with DMEM/20 FCS as a backup. Volume to the T25 culture 5 mL with DMEM/20% FCS (add 3.5 mL) Place all cultures in the incubator.