I am wish to culture it T cells from PBMC, hence I wish to remove monocytes from PBMC by attachment to a surface. I wish to know what sort of surface/plasticware/glassware can be used to remove monocytes by attachment method and what are the other variables or factor like Media or FBS affect this step. I wish to know about it in detail. Thank you for your help.
Monocytes will readily adhere to standard plastic tissue culture plates. I recommend using large wells, 12-well or 24-well plates should be fine. Do not seed the cell suspension too densely otherwise there will be overcrowding and a chance you will not achieve maximal adhesion. Also consider a time course to assess the minimal amount of time required for this step - you don't want them hanging around too long together.
If you are interested in isolating just T-cells an alternative (and superior) method is to use immunomagnetic negative selection. Removing monocytes by adhesion will not be 100% effective, also you will still have B-cells in your suspension which may impact your end results. By not using a 100% pure population there is a higher chance of variability in your results.
Good luck!
I totally agree with Andrew Higham. In your first attempts you should chek purity by flow cytometry.
thank you guys for your valuable suggestions, I am just doing my preliminary expt., but when I go on with my the actual aim. I am planning to isolate the T cells with anti@CD3/CD28 beads.
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