How to make serum for tissue culture?

John Ladasky jladasky at pmgm.Stanford.EDU
Mon Feb 16 15:07:18 EST 1998


Hi, folks,

	I'm trolling this through sci.med in the hopes of picking up 
some M.D. readers who have hematology experiemce but who don't ordinar-
ily read the bionet newsgroups.  Similarly for bionet.immunology.  How-
ever, I have set the followups back to bionet.cellbiol, which I think
is the most appropriate newsgroup for this rather technical discussion.

	I've run into an interesting problem.  I am working with an an-
tibody to the class I MHC molecule.  I am studying class I MHC in a
species of New World Monkey; some individuals react to this antibody,
and some of them do not.  The antibody recognizes an epitope that spans
across the class I heavy chain and beta-2 microglobulin.  I have identi-
fied an amino acid polymorphism in beta-2m that is responsible for the
antibody reactivity.  

	You may recall that class I heavy chains are membrane-anchored
molecules, but beta-2m is not.  So class I heavy chains can lose their
endogenous beta-2m in cell culture, and pick up the beta-2m from the cul-
ture medium.  Bovine serum contains beta-2m -- and, unfortunately, that
beta-2m contains the polymorphism that creates reactivity to my antibody.
So when I want to do flow cytometry analyses or immunoprecipitations,
this is a nuisance.

	That way that I have been getting around this problem for now is
to wash my cells 3-4 days in advance of my experiments, and then let them
sit in serum-free medium.  This gets rid of the unwanted background, but
it also kills roughly half of the cells -- this creates a flow cytometry
headache, as the machines clog with dead cells.  Ideally, what I would 
like to do is to prepare a serum that preserves cell viability, but does
not contain an intruding beta-2m molecule.  

	I have been experimenting with the monkey blood that I have been
receiving.  The animals are not living on-site at Stanford -- I get small
vials of blood shipped by Federal Express.  The animals are quite small,
and they are bled with fine-gauge needles into 3 ml Vacutainer tubes.  On
the basis of my experience with human blood and with chimp blood which
also has been shipped, these monkey blood samples have an unusual amount
of hemolysis.  Even after I centrifuge the blood for 30 minutes at 400 g,
the serum layer is noticeably pink, rather than the yellow color I'm used
to seeing.  Presumably this pink color is due to hemoglobin from lysed
red blood cells.  An M.D. postdoc in the lab does not think that this is
a problem specific to the monkey blood.  He says that drawing blood with
small-gauge needles, even from humans, generates a significant level of 
hemolysis. 

	I decided to try to prepare some serum anyway by pooling the sam-
ples from the animals that were negative for antibody.  I have a protocol
from R. Ian Freshney's _Culture_of_Animal_Cells:_A_Manual_of_Basic_Tech-
nique_ (Alan R. Liss Inc., 1987) which is rather inappropriate, since it
is designed for isolating serum in the liter range from whole cows.  Also,
it says nothing about heat-inactivating the serum -- my understanding is
that 56 degrees C for 1 hour destroys the complement proteins which can 
lyse your cells.  Anyway, I tried various things with the small quantities
of serum I have.  0.22 micron syringe filters clogged quickly -- maybe 
due to the excess hemoglobin in the serum -- and I lost yield.  And 
whether I tried my one-hour heat-inactivation step before or after filter-
ing, I got this cooked-egg effect -- again, probably due to the excess of
hemoglobin protein.  There was still some liquid left in the preparations.
I have salvaged what I can by centrifuging the filtered, heat-treated
serum for five minutes at 16,000 g.  What I have left is clear, but since
I gummed up so much of the protein in the heat-inactivation step, I wonder
if I have anything useful left...

	Any advice would be appreciated!

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



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