Manuscript Sections Organization

Methods

Hybridoma Protocol

  • Note: you only want to mention any differences that were made to the protocol!

  • Obtain the 25 cm2 flask with cells that have been passed in Medium A two days before fusion. Transfer the parental myeloma cells to a 50 mL conical tube and centrifuge at 300 x g for 10 minutes at room temperature. Discard the supernatant and add 30 mL of ClonaCellTM-HY Medium B. Centrifuge the tube at 300 x g for another 10 minutes at room temperature and discard the supernatant. Repeat twice for a total of three washes. Resuspend the pellet in 25 mL of Medium B. Count the number of live cells following step 2.6 and record the viability percentage. Calculate the volume of cell suspension that contains 1 × 107 viable cells. Keep cells at room temperature until fusion.

  • Gather a 50 mL conical tube, 75 cm nylon mesh, a syringe plunger, and Medium B. Sterilize this equipment before putting it in the laminar flow hood. Remove the lid of the conical tube and place the nylon mesh over the mouth of the tube. Center a spleen on the mesh and use the syringe plunger to gently disaggregate splenocytes. Wash the splenocytes with 30 mL of Medium B. Centrifuge the tube at 400 x g for 10 minutes at room temperature. Remove the supernatant with a pipette and discard into the hazardous waste. Repeat for a total of three washes. Resuspend in 25 mL of Medium B. To count the splenocytes, follow the same steps outlined in 2.6 and calculate the volume of cell suspension that contains 5.0 × 107 cells. Keep the cells at room temperature until fusion.

  • Place ClonaCellTM-HY Medium A, ClonaCellTM-HY Medium B, ClonaCellTM-HY Medium C and ClonaCellTM-HY PEG in a water bath set to 37oC. Add 1 × 107 parental myeloma cells and 5.0 × 107 splenocytes to a 50 mL conical tube. Centrifuge this solution at 400 × g for 10 minutes. Carefully discard the supernatant using a pipette and do not disturb the pellet. Disrupt the pellet by gently flicking the tube. Obtain ClonaCellTM-HY PEG from the water bath. Slowly add 650 µL dropwise to the pellet using a P1000 for a duration of one minute and do not mix. Once this volume is added, combine the cells with ClonaCellTM-HY PEG by swirling the solution for a minute. Add 4 mL of Medium B to the fusion mixture and stir for four minutes. Add 10 mL of Medium B and then incubate at 37oC for 15 minutes. Slowly add 30 mL of Medium A and centrifuge the tube at 400 x g for 7 minutes. Discard the supernatant using a pipette and do not disturb the pellet. Slowly add 20 mL of Medium A to resuspend the pellet and centrifuge with the same settings stated prior. Discard the supernatant and resuspend the pellet in 10 mL of Medium C. Transfer this volume into a clean, sterilized 75 cm2 flask containing 20 mL of Medium C. Incubate the flask for 16-24 hours in 5% CO2 and 37oC.

  • Bring ClonaCellTM-HY Medium D to room temperature. Transfer fused cell suspension from the 75 cm2 flask into a 50 mL conical tube and centrifuge at 400 x g for 10 minutes at room temperature. Remove the supernatant using a pipette and resuspend the cells in Medium C to a total volume of 10 mL. Transfer 10 mL of cell suspension into 90 mL of Medium D. Mix thoroughly and incubate for 15 minutes at room temperature. Transfer this solution into a v-boat and use a multi-channel pipette to transfer (__) µL into rows of a 96-well plate. Incubate the plate at 37oC and 5% CO2 for 10-14 days. Do not disturb the plates during this time.