Sample： splenocytes of mouse model
I have been using MACS negative isolation method for 1.5 years, and it worked fine.
usually the yield will be ~1M/spleen, but in recent half year, I kept getting 1/2 or even less of the yield for each spleen.
I have tested the PH of MACS buffer, different boxes of columns and a new vial of antibodies, which didn't make any difference.
I also tested the viability of target cells in each step using Flow: proving the target cells are good.
And I collected the cells remained in the column and did surface markers staining of my target cells in those elute (microbeads-bind population) : suggesting the target cells are still there and wasn't extracted out.
Anyone knows how to improve the yield??
Thank you so much !
Usually nylon-wool column equilibrated with FBS/ FCS [3%, v/v] works fine for separation of mouse or human T-cells from spleenocytes which as excluded by the column. The B-cell remain attached with nylon-wool which may be subsequently eluted using cold RPMI-1640 medium.
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