Hello all:
I am interested in isolating pig monocytes from whole blood. I will do so by Ficoll followed by MACS for CD14 marker.
My 2 questions are: does anyone know the best culture media for maintaining/proliferating adult monocytes, even if not specific for pigs? Also, I want to co-culture these monocytes with plasma I obtained before (kept at -80 since sampling) and evaluate expression of HLA-DR by flow cytometry. Does anyone have an idea of the time I should let the monocytes be co-cultured with the plasma? Should I do step-wise increases in the percentage (vol/vol) of plasma I should add to the culture media prior to deciding how much to add? I dont have a lot of plasma sample from each of the studied animals, so I really need to be cautious with how much I use. I am thinking of using 96 well microplates for culturing so I need little volume from plasmas for the co-cultures.
Any advise will be greatly appreciated.
Thanks a lot.
Hi,
Pig monocytes do represent a quit big group of cells which are characterized by different types of surface markers including CD14. Subgroups of monocytes including antigen-presenting cells exert different functions. I am wondering about the purpose of your cell isolation ?
Have a nice weekend !
Regards,
andrea
You might look at this paper.
http://www.ncbi.nlm.nih.gov/pubmed/9692868
Thank you all for your help. With regard to Dr. Geiss´s question, I am looking at the effect of different modes of mechanical ventilation over monocyte antigen-presenting capacity, as measured by HLA-DR (or the homologue SLA-DR). Patients with lung failure that are put on mechanical ventilation may be at increased risk for developing subsequent nosocomial infections and I want to evaluate the ability of mechanical ventilation-induced inflammation to induce immunoparalysis in peripheral monocytes. I want to isolate peripheral monocytes from healthy pigs and subsequently co-culture them with plasma I have obtained from pigs treated with different ventilatory modalities.
Do you think isolating based on CD-14 would be inappropriate?
If you check the articles by David Hume on porcine macrophages or Artur Summerfield, you will find the best combination of mAbs to identify porcine monocytes - for example CD172a might be a better marker for sorting. Do you want to sort by FACS or MACS? In addition, please keep in mind that peripheral monocytes in pigs behave different to alveolar macrophages (which can easily be isolated in pigs), so might be worth considering these for your experiment. FCS works fine for pig cells, and in combination with RPMI, Pen?Strep and 2-ME, you have a good medium. However, the cells won't proliferate, you can only maintain them. If you add CSF-1 into the medium, the cells are quite homogenious at the end. Cheapest source of that if supernatant of murine L929 cells, which contains CSF1, and murine CSF1 cross-reacts with the porcine CSF1R. Up to 10% of L929 sup is fine, but you may want to titrate.
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