Editing Hybridoma production (section)

== <br/>Fusion ==

1. Spin down&nbsp;mixed cells 900-1000rpm 5 min.<br/><br/>2. Wash cells with 25ml Hyb-SFM medium (no additives) or serum-free DMEM&nbsp;and centrifuge

(just do one wash only; do not resuspend)

3. Loosen pellet by finger-flicking. Aim for a slurry of cells; do not want chunks or&nbsp;pelleted cells.

4. Slowly add 1.5ml PEG per 3x10<sup>8</sup>&nbsp;&nbsp;mixed cells. (Rick's protocol, 1 ml PEG for spleen cells from one mouse)

*(Do at room temperature – do not need to keep in 37C waterbath.

*Dispense PEG slowly&nbsp; along sides of tube. Does not need to be 1ml/min slow, just&nbsp;slow enough to keep from blasting cells.

*Do not touch cell/PEG slurry with pipette. After dispensing PEG, mix by&nbsp;swirling tube.)

5. Incubate for 1 minute at 37 oC, Rick's protocol: 40&nbsp;seconds

6. Add very slowly 1 ml of serum-free DMEM alone sides of tube

7. Incubate at 37 oC for 30 seconds

8, Add slowly 2 ml of serum-free DMEM&nbsp;to the cells

9, Incubate 22 seconds at&nbsp;&nbsp;37 oC.

10, Add 4 ml of&nbsp;serum-free DMEM&nbsp;slowly to the cells

11,&nbsp;Incubate 22 seconds at&nbsp;&nbsp;37 oC.

12,&nbsp;Add&nbsp;8 ml of&nbsp;serum-free DMEM (no need to be very&nbsp;slowly this time) to the cells

13,&nbsp;Incubate 22 seconds at&nbsp;&nbsp;37 oC.

14,&nbsp;Add&nbsp;16 ml of&nbsp;serum-free DMEM (no need to beslow this time) to the cells

15,&nbsp;&nbsp;Incubate 22 seconds at&nbsp;&nbsp;37 oC.

Alternatively use this method:

Add a total of 20ml Hyb-SFM. (use two 10ml pipettes).<br/>1 ml in first minute<br/>3 ml in second minute<br/>(Dispense as close to dropwise as possible. Times should be treated as bare minimums – if it takes longer, so be it.)<br/>16ml in third minute

<br/>16. Centrifuge&nbsp;900-1000rpm, 5 min.

17, Remove medium

18, Add 25 ml DMEM with 15% serum, transfer to sterilze reservoir

19. Add medium (DMEM with 15% serum) to 100 ml.

20. Aliquot 100 ul to ten&nbsp;96-well plates.

or&nbsp;use tissue-culture treated 24 well plates, 10<sup>6</sup>&nbsp;&nbsp;total cells/well,&nbsp;&nbsp;&nbsp;2ml cell suspension per well

21. Culture cell in CO2 incubator