It is always safe to add anti-mycoplama reagents in your culture medium whether you are dealing with human materials or animal samples. When we get synovial biopsy, what we do is as follows 1) first treat biopsy materials with collagenase to get single cell suspension. 2) After about 7 days culture, what you see is either macrophages or fibroblasts in adherent population, actually mixture of those cells. You can discard floating cells. If you flush the plate with fresh medium, you can eliminate some of macrophages. By treating those cells with mild trypsin or EDTA containing medium, you can recover single cell suspension. Re-plate those cells. If you repeat this cycle 2 or 3 times, what you can get is fibroblasts. Purity can be determined by FACS.

Thanks for replying sir. I have few minor (or may be the most important) questions...How should we bring (or store) the tissue from doctor after surgery??should we take it in RNA later (as for maintaining RNA integrity) or which medium is appropriate? In your reply you have mentioned after seven days culture, do you mean it for the first passage only?? Should we add anti-mycoplasma (e.g Cellmaxin) in the medium just when we start culturing? Please reply.

I recommend you to bring human synovial tissues from Operation room to Lab, right away. No freezing is recommended. I usually add anti-mycoplasma reagent in the first passage only. Usually for the first 7 days culture, we change medium at day 3 or 4 depending on the proliferation of cells. Both medium should contain anti-mycoplamsa reagents. If you are interested in gene expression study, split synovial tissues into tow peaces; one for culture and one for RNA extraction. Even for RNA extraction I recommend that you do RNA extraction without freezing.

I have cultured the RASF from patient biopsy obtained. Now i can see a lot of granules surrounding the fibroblast in the culture flask, I want to know that are these lymphocytes/ macrophages or is it some contamination. while taking the picture on a computer it seems to be moving so i am worried if its a bacterial contamination. please reply.