Freezing solution: 90% FCS + 10% DMSO, ice cold.

1) Spin down E7 cells (E6 minimum) at 1200 rpm for 5 min.

2) Aspirate medium.

3) Resuspend in 1 ml of ice cold freezing solution.

4) Transfer vial to an insulated freezing box and place at -70 C for at least 1 hr. (could be for a couple of days).

5) Transfer the vial from the -70 C to the liquid nitrogen tank and log the entry in the freezer log.

Thawing cells:

1) Take vial out of liquid nitrogen tank and thaw it immediately in a 37 C bath (about 1 min).

2) When there is still a small piece of ice left, dilute the cells by transfering them into a conical tube containing 10 ml of the growth medium at 37 C.

3) Spin at 1200 rpm for 5 min.

4) Aspirate medium and resuspend cells in 5 ml of medium, in a 25 cm2 flask.

Cell Fusion and Selection

Solutions:

IMDM

IMDM complete + 15% MCM.

PBS

PEG 50% ( w/v ): PEG 4000 ( 50% ) from Boehringer Manheim 1 243 268 Aminopterin: Sigma, A 5159 (50X )

1) Prepare a T-150 flask with 170 ml of IMDM complete with MCM instead of PCM (= IMDM - m ), and keep it in the incubator for fused cells.

2) Isolation of spleen cells :

a) Sacrifice mouse by cervical dislocation. Immerse mouse in 70% Ethanol.

b) Remove spleen ( on the left side ) and transfer into a small petri dish which contains IMDM at room temp. Clean fat from the spleen and transfer the spleen into an empty dish.

c) Using sharp tipped forceps, one end is punctured. A curved forceps is used to hold down the intact end, and the spleen is gently rubbed towards the opened end with another set of forceps. The cells from inside the spleen will ooze out with very little damage. Stop the process when you are left with a nearly empty, transparent skin. Collect the cells by rinsing with IMDM. Alternatively, pass the spleen through a sterile stainless steel grid (#60) to separate the cells and the stroma.

d) Transfer cell suspension to a 15ml conical tube and let the cell debris settle out (approx. 5 min.).

e) Remove the cell suspension (without disturbing the settled cell debris) and transfer to a 50 ml conical tube. Add an additional 30 ml of IMDM and pellet the cells at 1200 rpm for 5 - 10 min.

f) Aspirate the medium, resuspend pellet and wash again with 30 ml of IMDM. One immunized spleen has aprox. 108 cells. After this wash the cells are ready for the fusion.

3) Myeloma cell preparation: It is essential that the myelomas be free of debris, rounded and refractive under phase contrast, and that they are harvested in log or late log phase growth (between 3.5 and 9E5 cells/ml).

a) Thaw cells 7 days before scheduled fusion. Myeloma do not grow well after being in culture for more than a few weeks.

b) Make sure you refreeze cells for future use.

c) We have been using a ratio of 2 spleen cells : 1 myeloma cell. However, workers have been using a ratio from 1 to 10 spleen cells per myeloma successfuly. For one spleen we harvest 5x107 cells. It is advisable to do this spin at the same time that the second wash of the spleen cells is done.

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