synovial fluid - (Sep/10/2008 )
Has anyone of you tried separating mononuclear cells from synovial fluid? I am working with rheumatoid arthritis patient's synovial fluid and trying to isolate mononuclear cells from it using ficoll-hypaque method. My success rate is low ie of the twelve samples I have collected , I was able to clear interphase of mononuclear cells only from 4 samples. With this success rate, I find it difficult to proceed with my research work.
I am adopting the standard procedure of ficoll-hypaque ie I pellet down the cells in synovial fluid by spinning the fluid at 600g for 10 min at RT. The pellet is suspended in RPMI media in conical tube and carefully layered on Ficoll-hypaque. Teh tubes are spun at 1000g for 25 min at RT.
Kindly someone suggest me the steps I have to adopt for getting clear interphase layer. I will be thankful to you. Please help.
Is it just low recovery? Can you mix the fluid with RPMI first and layer the mixture? Or, concentrate the fluid with Minicon, mix with RPMI and try again? Do you have any idea as to number of cells/ml you are working with?
I can't think of low recovery as I have alluded in many papers that the mononuclear cells count is around 60 - 70% in Rheumatoid arthritis patient's synovial fluid, which can be efficiently isolated by using Ficoll-hypaque method. With my samples, I have recovered 1-2 X 10 ^ 6 cells /ml. When I spin the synovial fluid, the cell pellet is considerable.
I have tried mixing the synovial fluid with PBS and yesterday I was able to recover a thin layer of mononuclear cells. Also, I was able to see a flock of cells in the ficoll layer which are presumably granulocytes which are supposed to settle down with erythrocytes.
I would like to know how to get a clear interphase of mononuclear cells. Kindly help me with a solution.