How to make serum for tissue culture?

Guy Hermans ghermans at luc.ac.be
Wed Feb 18 02:23:16 EST 1998


In article <6ca69m$8v2 at pmgm.Stanford.EDU>, jladasky at pmgm.Stanford.EDU
(John Ladasky) wrote:

<snip>
> 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!

Hy John,

we culture human antigen-specific T-lymphocytes all the time. One big
nuisance is having to work in 'autologous medium', which is basically
RPMI1640 souped up with additives and ...autologous serum. So I might say
we have a lot of experience with these problems.

First of all: yes, fine gauge needles increase haemolysis dramatically,
due to simple mechanical stress on erythrocytes.

Second: the protocol for human serum is: 
1. Centrifuge the blood at about 2500 rpm (10' will do) in a benchtop centrifuge
2. Collect the supernatant (yellowish serum)
3. Filter through the following series of syringe minifilters:
     prefilter (Millipore white disc-type syringe filters)
     filter through 1 micrometer filter (Sartorius yellow syringe filters)
     filter through 0,22 micrometer needle-type filter (sterile), collect
in      sterile centrifuge tube

4. Inactivate: preheat waterbath to 56°C, place centrifuge tube with serum
in it and incubate for 30 minutes. Unlike foetal calf serum, human serum
turn from beatifully clear gold-yellow to opaque off-white/yellow, due to
precipitation.

5. Cool down the tube; straight from 56°C into the fridge for a few hours.

6. Centrifuge the tube at 2500 rpm in benchtop centrifuge (10').

7. Collect superantant, and store in aliquots at -20°C or below. Can be
kept for months at this temp. Do not thaw and freeze down again for more
than once.

We use 10% inactivated serum for most purposes, but transformed cells can
be grown in 5%. Results from various cell types and sera vary; check it
out before relying on a standard protocol. And that's the point where I
end this protocol ;-)


Good luck,

Guy

--------------------------------------------
Guy Hermans, PhD student
MS research Unit             Tel 0032 (0) 11/26.92.07
Dr. L. Willems-Institute     Fax 0032 (0) 11/26.92.09
University Campus            E-mail ghermans at luc.ac.be
B-3590 Diepenbeek
Belgium
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