Polyethylene Glycol (PEG) Mediated T Cell Fusion

1 post / 0 new
Tony Rook
Tony Rook's picture
Polyethylene Glycol (PEG) Mediated T Cell Fusion

Please find the link for the following protocol:

Polyethylene Glycol (PEG) Mediated T Cell Fusion

Link Reference:
http://www.uchsc.edu/misc/diabetes/derc/protocols/immunologyprotocols.pdf

The fusion parent of choice is the TCR abvariant of BW5147, to this are fused T cells from a line or clone reactive to the antigen of interest. Typically the T cells are obtained by harvesting the inguinal and periaortic lymph nodes of mice primed to the antigen of interest with CFA/antigen. The lymph nodes are disrupted into cell suspensions and cultured for 4
days in Clicks medium with 0.5% NMS and then placed in IL-2 for 3 days to expand. T cell clones and lines can be successfully fused following an analogous schedule, in this case the clone or line is set up in a standard re-stimulation flask with 5.0 X 105 T cells, 2.0 X 107 irradiated NOD spleen cells in 20 ml DMEM/10% FBS with 50U/ml IL-2. The cultures are incubated for four days at which point they are subjected to Lympholyte M separation and culture for 3 days in 50 U/ml IL-2, the cells are then counted and washed as below.

Hybrid cells are selected by culturing the fused cells in hypoxanthine/aminopterin/thymidine (HAT). The aminopterin component of HAT inhibits a key enzyme in purine and thymine
biosynthesis, cells in culture can survive this inhibition due to the presence of an alternate pathway and the exogenously added hypoxanthine and thymidine, a key enzyme in this
pathway is not functional in BW5147 and this cell line dies in the presence of HAT. The presence of a normal enzyme encoded in chromosomes of the normal T cell component of the hybrids allows them to survive in HAT.

Materials

200 ml of serum free DMEM @ 37
200 ml of BSS @ 37
200 ml DMEM/10% FBS @ 37
3 1 ml aliquots of 50% PEG
50 ml polypropylene tube
0.4 μ syringe filter
15 ml tube
50 ml tubes
sterile plugged pasteur pipettes
25 ml pipette
10 ml syringe
Prepare all in advance and filter

Procedure

Preparation of 50% PEG solution
1. Place PEG (MW=1500) (usually in a 50 ml polypropylene tube) in boiling water,
2. Once the PEG has melted weigh out 2 - 3 grams noting weight to second decimal,
3. Quickly add 1.0 ml / gram of serum free DMEM, mix and sterilize by filtration through a 0.4 μ syringe filter into a 15 ml tube,
4. Remove approximately 3 ml and place approximately 1 ml in each of 3 sterile 15 ml tubes.

T cell fusion
1. Count BW5147s and remove 3.0 X 107 viable cells, pellet in 50 ml tubes and pool
into one tube, pellet and leave at room temperature
2. Count T cells to be fused and remove 2.5 X 107 to 5.0 X 107 cells, pool pellets and add to pellet of BW5147 cells, bring volume to 50 ml with DMEM/HEPES and wash, resuspend cells and repeat wash two more times
3. After the final pelleting aspirate off the DMEM and spin tube again for 5 min to get last traces of medium, aspirate carefully and get the pellet as dry as possible, during this last spin add about 1600 ml 39 40oC tap water to a clean 2 L beaker and tape one of the aliquots of PEG to the beaker with the PEG solution below the surface of the water
4. Hold the tube with the cells approximately horizontal and tap firmly against the lip of the hood and rotate the tube to distribute the pellet of cells over as much surface area of the conical bottom as possible. Once the pellet has been distributed, loosen the cap and rest the tube in the water. Draw the PEG solution into a sterile plugged pasteur pipette, remove the cap from the tube of cells and place it in a water filled beaker in the back of the hood. Dribble the PEG over the sheet of cells from a
height of the rim of the tube over a period of approximately 45 seconds shaking the tube and gently tapping against the side of the beaker while keeping the lower part of the tube in the water, after all of the PEG has been added replace the cap and place the tube in the water bath while a 10 ml syringe is filled to the limit (approx. 15 ml) with 37 oC DMEM/HEPES;
5. 45 seconds after the PEG addition was completed begin to dilute out the PEG by adding the DMEM drop-wise and gently swirling to mix the PEG with the DMEM, after all 15 ml have been added, gently fill the tube with DMEM using a 25 ml pipette, use care to minimize the shear forces due the fragile nature of the fusion products at this time, incubate the tube at 37 oC for 10 min and pellet the cells (10min @1000 rpm), aspirate off the DMEM and loosen pellet by gently tapping on
the hood surface, gently add 25 ml DMEM/HEPES, pellet, loosen pellet by tapping and resuspend in approximately 40 mls of DMEM/10% FBS
6. To minimize shear forces that might disrupt hybrids it is essential that all pipetting be done gently without trapping the tip of the pipette on the bottom of the tube. The cells should be distributed into 4 96 well plates @ 0.1 ml of cell suspension per well
7. Approximately 24 hours after the fusion add the HAT supplement to the plates by preparing 40 - 50 ml of DMEM/10% FBS with 2X the final concentration of HAT
8. If the fusion is a success, hybrid growth will be apparent at day 4 - 5 by examination with an inverted scope, the hybrids will be ready for transfer to flasks approximately 10 - 14 day after the fusion.