need help

simon cross pcxsc at
Tue Feb 29 11:07:11 EST 2000

The method most people reference is from Kohler and Milstein, Nature,
1975, 256, 495-497 but this is quite old and you will probably need to
check a more recent publication. The method I use is as follows:

1) Disect mouse to get spleen - use 3 different sets of
scissors/tweezers to cut outer skin, inner membrane, and finally the
spleen itself to avoid contamination. Be VERY careful not to pierce the

2) Place spleen in 5ml prewarmed RPMI medium and tease apart to get cell
suspension.Try to remove large clumps of tissue.Count the cell density
-  should be about 10^7 or 10^8 cells total.

3) Take myelomas that have been growing healthily for at least 1 week,
count them and add to splenocytes (10 Spl : 1 Mye). Pellet mixture and
remove supernatant.

4) Agitate the pellet away from the sides of the tube and add 0.1ml PEG
1200 (prewarmed) over 1 minute with gentle shaking, then gently

5) Add 1ml RPMI serum free medium over 1min, with shaking then
resuspend. Then add 10ml medium over 5min.

6) Pellet at 1400rpm and discard supernatant. Resuspend fragile cells in
25ml HMT medium (15% FCS and 10% Hybridoma Enhancing Supplement) and
plate out into 96 well plates (I use about 6). Top up wells with more
HMT/HES and leave for about 10 days - you will need to replace medium at
7 days.

This method works for me - just be careful with the Hybridoma Enhancing
Supplement, I have had big problems with cells becoming 'addicted' to
it. Don't use it after your 7 day feed, except to clone the cells. I
hope this is of some use.

Simon Cross
School of Chemistry
University of Nottingham

Gautam Sondarva wrote:

> Hello!!
> I am looking for assistance in making spleenocytes from mice.
> How do I go about doing it?
> I shall be happy if someone can give me directions as to where
> I can find a method OR if someone can be kind enough to send
> me a step-by-step protocol for the same.
> I am aiming at immortalizing these spleenocytes, hence would be
> happy if someone can give me additional information for the same.
> OR atleast a method which will allow me to maintain these cells for
> a short time in tissue culture.
> Thanking you all in advance
> Gautam
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