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Why do I not have any cells left in my positive panning plate after transferring from the negative panning plate during immunopanning? I am trying to purify retinal ganglion cells from postnatal rats and use a Thy1.1 antibody harvested from a hybridoma cell line supernatant in the positive panning plate.

Following transfer of the cell suspension from the negative to the positive panning plate there are no cells in the pre-trypsinization supernatant, nothing stuck to the panning plate, and no cells in the post-trypsinization supernatant so I have no idea where they could have gone. Checking the negative panning plates however shows that there are many cells stuck to those plates, so could my RGCs have been lost at some point during the transfer and when?

Thanks v much in advance for your help.

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Did you check the actual transfer material for cells before you added it to the positive plate? It may be they all stuck to the negative plate... –  MattDMo Aug 20 '13 at 21:46
    
Is immunopanning better than labelling with an antibody and sorting by FACS? –  leonardo Oct 20 '13 at 22:41
    
Or using MACS for sorting? –  Chris Dec 19 '13 at 17:32
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1 Answer

Immunopanning in my experience takes an awful lot of optimization, but is worth it when it works. I would plan, if you have the resources, to sac rats just for assay optimization before you even begin to try to get real data out (assuming your planning for a steady stream of primary cells).

That said, there are some real easy checks you can try do first. Try to get a rough cell count on what you put on your negative plate (depending on your prep steps, that can actually be a bit tricky), and then count the cells again as you transfer the cell suspension to the positive plates. If you don’t have any cells left, or you have very little cells left, then you might be losing all your population on your negative plate.

If you do have cells in suspension, I would start to question your positive plate. Are you making them yourself or are you buying them coated? Which rat strain are you using? Perhaps you need to titer your antibody concentration on the plate. There are really a whole lot of steps in immunopanning that can be optimized.

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