Hello again.
In continuation of my problems concerning growth of hybridoma cells,
I will try to give a more full story of what we actually did. This
is due to the responses that I've got (and I would like to thank the
people who showed interest).
1) 10 e8 spleen cells and 5x10 e7 myeloma cells were mixed in 10 ml of
RPMI 1640 + L-Glutamin, and the cells were spun down.
2) 50% PEG 4000 (Gibco) were added to the pellet over 1 min. and the
cells were allowed to rest for 1 min.
3) Then 10 ml of RPMI 1640 + L-Glutamin were added and the cells were spun
down again.
4) Cells were resuspended in RPMI 1640 + L-Glutamin + 20% FCS + 10 % HECS
(Human endothelia cell supernatant). The cells were plated out in 96 wells
plates coated with collagen, 100 ul in each. and allowed to rest for 1 day.
5) Then 100 ul of The same medium as in 4) supplemented with 1xHAT and
Pen/strept. were added on top of the old medium. then rest for 1 day.
6) Half of the medium were then taken off and replaced with fresh medium
as in 5). This is continued until day 8.
7) on day 11 the cells are tested for antibody production and the positive
are transfered to 6 wells plates which is still coated with collagen. and
the medium is still RPMI 1640 + HAT. (The passage is with 0,1% trypsin)
8) 3 to 4 days later the cells are passaged. Again by 0,1% trypsin after which
the cells are collected in RPMI 1640 + 20% FCS and at this stage we change
from HAT to HT as supplement, and spun down. They are then resuspended in
in the same medium RPMI 1640 + L-Glutamin + 20 % FCS + 10% HECS + 1xHT.
It is then our problems arise. The cells attach to the plates but they will
no longer proliferate.
I hope this helps some of you more experienced people out there, in relating
to our problem and maybe give some suggestion how to overcome the problems.
In advance I thank for the interest.
Peter Kristensen
Aarhus University
Department of Chemistry
Division of Biostructural Chemistry
Denmark
E-mail : Brian at bio.aau.dk
