How to make serum for tissue culture?

John Ladasky jladasky at pmgm.Stanford.EDU
Mon Feb 16 22:53:21 EST 1998


Hello, Larry,

	For some reason this followup did not go back to bionet.cellbiol.
Oh well, if nobody is objecting to this thread in bionet.immunology, where
you responded to it, I guess we'll keep it there for a while.  You've given
me some interesting ideas, but it's clear that I have to clarify my earlier
statements.

In article <Pine.GSO.3.96.980216145737.17907A-100000 at catbert.ucdavis.edu>,
L.A. Pike-Nobile <ez063669 at mailbox.ucdavis.edu> wrote:
>John,
>
>Perhaps I'm missing something here, but if you heat-inactivate your serum
>(56oC, 1 hour) doesn't that cook the remaining B2m and make it unavailable
>for complexing with MHC I? Particularly since you have an antibody which
>recognizes an epitope spanning the two molecules, I'd think heating (and
>pehaps addition of a little 2ME) would eliminate your problem by
>eliminating the ability of B2m to complex with the a3 domain of MHC I. 
>
>Or am I missing something fundamental about small globular proteins? 

	This is an interesting idea -- maybe I *could* use ordinary fetal 
calf serum if I reduce the disulfide bonds when I heat-inactivate it.  It
is my understanding that beta-2m, if not reduced, doesn't irreversibly
unfold at 56 degrees.  It's just a single immunoglobulin domain.  But
would I also destroy some critical growth factors by doing this?  Isn't
commercial fetal calf serum heat-inactivated?  Do they reduce it while per-
forming the heat inactivation?  As I said, a good protocol would be help-
ful.

	That being said, let me remind you that I'm experimenting with 
serum from the monkeys that I know are negative for antibody reactivity.
So I don't have to denature the beta-2m.  If a small amount of MAb-nega-
tive beta-2m binds to my cells and reduces specific fluoresence by, say,
10%, this is far less of a problem than if some MAb-positive beta-2m 
comes in, increasing my negative signal to 10-20 times background levels.

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky



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