PROTOCOL
- prepare the culture of myeloma cells and maintain the concentration of the cells at the level of less than 10^6 cells/ml (IMDM + 10% FCS/FBS). Just before the fusion wash 3 times in IMDM (serum free!), 1200 rpm, 5min, 37ѓC. Count cells.
- sacrifice the mouse and dissect the spleen.
- tease the spleen in ice-cold IMDM (serum free!); pass through the nylon filter; wash 3 times in IMDM (serum free!), 1200 rpm, 5min, 37ѓC. Count cells.
- mix 10^8 spleen cells (in 25 ml of IMDM [serum free]) with 2x10^7 myeloma cells (in 25 ml of IMDM [serum free]) in Falcon-50 ml conical tube.
- centrifuge at 1200 rpm, 5 min, 37ѓC.
- aspirate the supernatant (SN) with a Pasteur pipette (all till the last drop!!!)
- gently break the pellet by tapping of the bottom of the tube.
- place the tube on 37ѓC water bath; add slowly, dop by drop 1 ml of pre-warmed (37ѓC) 50% PEG 1500 (polyethylene glycol, Roche, Germany) continously stirring the cells with the pipette tip (this procedure lasts for a period over 1 min)
- add 1 ml of pre-warmed to 37ѓC IMDM continously stirring the mixture (this procedure lasts for a period over 1 min).
- add 3 ml of pre-warmed to 37ѓC IMDM continously stirring the mixture (this procedure lasts for a period over 3 min).
- add 10 ml of pre-warmed to 37ѓC IMDM continously stirring the mixture (this procedure lasts for a period over 1-2 min).
- let to stay for 5 min on the 37ѓC water bath.
- centrifuge at 1200 rpm, 5 min, 37ѓC; aspirate the SN.
- resuspend the cells in IMDM-FCS(10%v/v) - HTx100 (1%v/v) - AJ(5%v/v) - IL-6, 0.5x10^6 cells/ml (calculation figured out from the number of spleen cells the fusion was started from); let to stay in incubator, 37ѓC, pCO2 7% for 2 hours in a big flasc.
- add aminopterin; distribute on 96-wells cell culture plates (Nunc) (200µl/well); place in incubator (37ѓC, pCO2 7%) enveloped in aluminium paper.
- check the cultures 1-2-7 days after the fusion.
- on day 7-10 change the culture medium.
- test clones when thay occupy 25% of the bottom surface of the well.
- IMDM (Iscove's Modified Dulbecco's Medium with 25 mM Hepes/with L-Glutamine) BioWhittaker Europe Cat.: BE12-722F
- IMDM-FCS(10%v/v)-HTx100(1%v/v)-AJ(5%v/v)-IL-6 (100 U/ml)
- IMDM - FCS(10%v/v) - HTx100(1%v/v) - AJ(5%v/v) - IL 6 (100 U/ml) - aminopterine x100 (1% v/v)
- HT (hypoxantine-thymidine)x100, 0.22 (or 0.45) µm-filtered, store at -20ѓC = hypoxantine solution, 50 ml + thymidine solution, 50 ml + ddH2O up to 250 ml
- hypoxantine solution = hypoxantine, 340 mg + ddH2O up to 50 ml + NaOH 1 N, 3 ml -> heat to 37ѓC
- thymidine solution = thymidine (2'-desoxy-thymidine), 96 mg + ddH2O up to 50 ml
- AJ (from "ajouter" , to add, local slang), 3 L, 0.22 (or 0.45) µm-filtered, store at -20ѓC = Asp-Arg x100, 1 L + Gluta x 50, 2 L + mercaptoethanol, 350 µl
- Asp-Arg x100, 1 L, pH 6.0 = L-Asparagine, 3.6 g (0.24 mM) + L-Arginine (0.55 mM), 11.6 g + HCl, 0.5N, 100 ml -> heat to 37ѓC, add ddH2O up to 1 L
- Gluta x50, 1 L, pH 6.0 = glutamine, 10.8 g (1.48 mM) + HCl, 0.5N, 100 ml -> heat to 37ѓC, add ddH2O up to 1 L
- aminopterine x100, 0.22-0.45 µm-filtered, store at -20ѓC = aminopterine, 4.4 mg (3.8µM)+ ddH2O, 50 ml + NaOH, 1N, 100 µl-> heat to 37ѓC, add ddH2O up to 250 ml
