Hybridoma protocol



Hybridoma protocol

(Peter Morganelli, with addendia)

Materials:

Water bath set to 39-40ºC

96-well cell culture plates with all of the outer wells filled with PBS (Usually about 6)

50 ml polypropylene centrifuge tubes

Sterilize:

4 pairs of jeweler’s type fine forceps

2 pairs of ordinary fine-pointed forceps

2 pairs of fine pointed scissors

one pair medium point scissors

25 ml beakers (3)

2 containers 9” Pasteur pipets

surgical drape

Media (recipes at end)

70 % ETOH

PBS

One bottle complete medium

SF medium without additives

50% PEG (Boehringer-Mannheim)

50X HAT

50X HT

Method

1. pre-warm PEG and all media to 39ºC

2. In the hood, set up three sterile 25 ml beakers containing 10 ml sterile SF media (does not have to be warm)

3. Sacrifice the mouse by CO2, followed by cervical dislocation

4. Immediately soak the abdomen in ethanol.

5. Place the mouse on its back on the sterile drape

6. Using sterile technique, with the fine-point forceps and medium scissors, cut the skin up the midline, then cut two flaps, pulling them back as you cut to preserve the sterility of the abdomen. Dip the instruments in ethanol. Cut the abdominal cavity wall in the same way, pulling the layers back as you cut to avoid contaminating the abdominal contents. With a fresh set of instruments (the fine ones) remove the spleen- cut it away from the faschia, removing as much of the attached fat as possible, and place immediately into a 50 ml centrifuge tube containing SF medium on ice

7. Exsanguinate the mouse by cardiac puncture- this blood will provide the polyclonal serum used as a control during screening

8. Dispose of the mouse. Take the spleen upstairs to the hood.

9. Dip the spleen in series through the beakers of medium

10. Place spleen in an 80 mm tissue culture dish

11. Add ~ 10 ml of SF medium

12. Dissociate the splencytes by teasing apart with fine pointed forceps, then squash with the sterile end of a 5 cc syringe plunger.

13. Transfer the suspension to a 50 ml polypropylene tube, bring up to 50 ml with SF medium and pellet for 3 minutes at 400 x g.

14. Remove the supernatant (Use a 9” Pasteur pipette, attached to vacuum). Add 3 ml of cold lysing buffer.

15. Vortex intermittently for 2 minutes, keeping the cells on ice between mixing

16. Add 15 ml of SF medium

17. Pellet for 3 minutes at 400 x g. Discard sup.

18. Resuspend in 10 ml SF medium. Do cell count/trypan blue viability. Keep cells in water bath until use.

19. Harvest 100 ml of NS1 cells. Pellet, resuspend in 10 ml SF medium and determine cell count/viability

20. Combine NS1 cells and splenocytes in a 50 ml tube at a 3:5 ratio (NS1:splenocyte) (Multiply the number of splenocytes by 0.6 to determine how much NS1 to add.

21. Vortex for 2-3 seconds

22. Pellet at 200x g for 3 min. (Do not remove sup)

23. Warm cell pellet for 10 min at 39º C

24. Remove supernatant and gently loosen pellet. Do so that the cells do not splash up onto the sides of the tube

25. Add 1 ml of warmed PEG dropwise, over 1 minute with gentle mixing. This step should be done while the tube is kept warm in a beaker of warm water.

26. Add 1 ml of warmed SF medium over 1 minute; add 3 ml over the second minute; add 16 ml over the third minute. Bring up to 50 ml with SF medium

27. Pellet for 5 min at 400 x g (This step achieves fusion)

28. Remove supernatant carefully.

29. Add 10 ml of warmed SF gently (without resuspending) spin for 2 min at 200 x g.

30. Remove sup and gently resuspend cells in complete NS1 medium containing FBS, such that there are 1 x 106 splenocytes per ml (based on original number of splenocytes). If the splenocyte count was between 40-60 million total, it is acceptable to bring up to 50 ml total volume.

31. Add 100 ul of suspension to wells of 96-well flat bottomed plates. Incubate at 37º C and 10% CO2

32. The next day, add 100 (l per well of 2x HAT prepared in NS1 medium.

33. Hybrids are ready for screening 7-21 days later. Typically, most are prime by 10-12 days. The cells grow rapidly, and actively growing cells (once a colony is visible) need feeding (fourfold dilution with fresh medium) every few days. The cells are NOT adherent, so use care pulling off the spent media.

34. Once the well start to turn yellow, screen for antibody production. Generally you can take 50- 100 ul of media off of the well from a 96-well plate. You can screen by ELISA or by Western blot. Replace the media with fresh HAT medium. The cells will tolerate one to two changes of media in the tiny wells, but tend to die after that, so they need to be cycled up to larger (48-well plates) wells.

35. To scale-up, I generally put 1 ml into the larger well, then, with a pipetman, gently agitate the cells in the 96-well plate, suck up as much liquid as possible, and transfer to the 48-well plate. At this point, the cells should be named. The default is to use the original plate and well numbers (e.g. 3D7) Some people like to refill the well on the 96-well plate as insurance. If you have a lot of positive wells though, this may make keeping track of which colonies have been scaled up a lot more difficult.

36. It generally takes several days to a week for the cells to outgrow the next size well. Continue scale-up as the wells turn yellow (or the cells reach confluence). It’s a good idea to transfer a little bit of the spent medium to the next size well with each transfer, since it will contain growth factors the cells need.

37. By the time you have worked up to a 12-well plate, there are enough cells to freeze, a good idea, even with a potentially unstable hybridomas.

38. It generally takes about 3 months for the hybridomas to become stable. The cells are kept in HAT medium until they reach the 12-well stage, then are gradually changed to HT (Every one has their own timetable and method for this. Generally the media change needs to be completed before you subclone.

Notes:

1. The outer-edge wells of 96 well plates show significant evaporation after a week. It is more cost-effective to fill these well with sterile PBS or water to minimize evaporation throughout the plate, since anything that grows in these wells usually does not survive the evaporation anyway.

NS1 Medium

DMEM-high glucose + glutamine

3.2 mM Hepes

2 mM Sodium pyruvate

10 % FBS-hyclone

5 x 10-5 BME

50 ug/ml gentamycin

BME:

9 ml of stock/ 25 ml SF medium Filter sterilize = 100X

Lysing buffer

01. M Tris HCL,

0.83% NH4Cl

use best quality water

pH to 7.5

Filter sterilize

SF medium- as it comes

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