I'm trying to purify rat Schwann cells using the Brockes method. We are using anti-thy1.1 antibody (AbD Serotec) and rabbit complement (Calbiochem) to remove fibroblasts and purify Schwann cells. The method works pretty well but the rabbit complement is not sterile and we have had, despite filtration, several bacterial contaminations. We've then asked for Sigma complement and it is not sterile either. I would like to ask if anybody knows which complement is best, if there's any sterile and, if not, how do you proceed to sterilize it.
Thank you very much!
I cultured rat Schwann cells by modifying the Brockes method and found that cytosine arabinofuranoside treatment alone was satisfactory to achieve >90% purity (verified by by S100 immunostaining). I have a protocol if you're interested. Good luck!
I used the same rabbit serum complement from Calbiochem and we never had contamination problems. Maybe your contamination issues are coming from some other source.
Hi Laura...
I tested and changed everything to the point that my final test was to add the antibody to two plates with Schwann cells but only complement in one of them. Only the one with complement was contaminated. Finally, i grew 2 plates with just culture media, and added the distilled water used to reconstitute de complement powder to one plate and complement in the other. Just the one with complement got contaminated. So i'm pretty sure it is the complement. The only thing is that i will probably will have to change the filters to see if i can avoid that.
But, thank you anyway.
We use Rabbit Complement from Sigma, product number S7764 (http://www.sigmaaldrich.com/catalog/product/sigma/s7764). We reconstitute it with water according to instructions, and store it at -80C divided to aliquots, so we do not need to freeze-thaw it repetitively. We have not done any sterilization steps, and still, have not had any contamination problems. We use ABs (penicillin/streptomycin) normally in our culture medium.
Hiya guys could anyone please post the method they used in purifying their Schwann cells. I have the anti-thy 1 and the complement and that's about it. No idea about the concentration i should use for either. Everything I have read has given me different answers and no exact method for complement mediated cytotoxicity. I read about how I have to do a hemolytic assay for my complement from Sigma, but there is no mention of that on this forum. I would just like an idea of concentrations and what was done to the anti thy 1 and complement. Cheers
Hi Modinat Liadi, do you have acces to our paper:
Isolation, purification and expansion of myelination-competent, neonatal mouse Schwann cells. Honkanen H, Lahti O, Nissinen M, Myllylä RM, Kangas S, Päiväläinen S, Alanne MH, Peltonen S, Peltonen J, Heape AM. Eur J Neurosci. 2007 Aug;26(4):953-64.
There you should find the "recipes", dilutions and how to do it.
I know this discussion is quite old, but maybe I am lucky here. I just started culture of primary murine Schwann cells according to the protocol of Honkanen et al (already mentioned above). While in principle I am quite fine with the results, I just don't get rid of the fibroblasts. I have the impression that the complement-mediated cytolysis has barely any effect. I use the anti-mouse CD90 from Serotec (clone F7D5) and the complement from Calbiochem and proceed just as stated in the protocol. Has Anyone any ideas or tips?
One possible thing that might affect: Which mouse/murine strain are you using? Because Thy1 allele expression is strain-dependent in mice and rats. The antibody you use is anti-Thy1.2, and, depending on strain, anti-Thy1.1 might work better.
Thank you for the quick answer. I already checked that. I use C57Bl/6, so Thy1.2 should be right, shouldn't it?
Yes, that should be OK then. How soon after cell isolation you do the anti-Thy treatment? According to my experience, C57BL cells are not as fast-growing as CD1 cells (the method was originally optimized using that strain). You can try to do complement kill again after a couple of days, if the fibroblasts seem to persist regardless of the complement kill. Another thing that might help, is to do the complement kill a bit later (if the fibroblasts are not too efficiently taking over the plate). Alternatively you could try to use higher concentration of anti-Thy and/or adding more complement.
Thanks again. I did the treatment 4d after isolation and since I saw fibroblasts afterwards, I repeated it 2 days later. Do you think that besides increasing concentrations, it might also help to increase the incubation times during the cytolysis?
I just tried another round of cytolysis using 8 µg/mL of the antibody (doubling the concentration) and 300 µL of the complement (increase of 50%). No change at all.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies