I am doing single cell isolation from tumor mass (using collagenase D & DNAse I) for FACS analysis. I also have 1 step for MACS cell separation after collecting single cell suspension. However, in some treated group, the cells in tumor are not in good condition. So I am worry that the long time processing could harm the cells. Do you think is it good to fix fix single cells (1% PFA) before performing MACS cell separation?
Thank you ^^
Fixation with formaldehyde affects antigen motifs and some antibodies that work very well on unfixed cells will not work well with fixed cells (we add some experience with antibodies against CD3, CD4 and CD8 that really did not like fixation). That is the reason why in kits for intracellular staining (like the Fixation/Permeabilization Solution Kit from BD Biosciences) the cell surface staining is done before fixing the cells.
So I think that Patrik is right here and you should optimize your cell preparation before risking to decrease the efficiency of your magnetic sorting with fixation.
Good luck.
Dear Nguyen,
Do I understand it correctly that you want to digest tumor tissue and perform MACS on these samples? We are performing these kind of experiments on a regular basis. As compared to FACS analysis, we are reducing the time for digestion in collagenase I and II from 1 hr to about 40 minutes with regular pipetting to ensure complete digestion. We are using the sorted cells for both RNA and protein analyses, as well as short-term culturing. The majority of cells are in good enough condition. Of course for these experiments for live cell analyses, we cannot fix in PFA.
I suggest you try to reduce your time for digestion to improve cellular quality. If you anyway want to fix cells, make sure that your antigen is able to being recognized by the antibody in question.
Good luck with your experiments!
I've analysed fixed splenocytes (Fixation/Permeabilization Solution Kit, BD Biosciences) because I needed FoxP3+ cells, and It worked. Kit contains formaldehyde 4%.
However, I've no experience with tumor mass flow cytometry.
Good luck!
Fixation with formaldehyde affects antigen motifs and some antibodies that work very well on unfixed cells will not work well with fixed cells (we add some experience with antibodies against CD3, CD4 and CD8 that really did not like fixation). That is the reason why in kits for intracellular staining (like the Fixation/Permeabilization Solution Kit from BD Biosciences) the cell surface staining is done before fixing the cells.
So I think that Patrik is right here and you should optimize your cell preparation before risking to decrease the efficiency of your magnetic sorting with fixation.
Good luck.
From one side, fixation may preserve cell structure and antigens structure, but, as has benne mentioned in previous posts, some antibody very sensitive to long fixation. You may do very gentle fixation (1% formaldehyde) for short time to save cell structure, but have a less effect on antobody binding. But this time must be detremine only expreriemnatlly. Best wishes!
Would it be an alternative to isolate single cells from the tumor mass directly using laser microdissection? Then you would avoid possible adverse effects by collagenase digestion and fixation.
http://www.molecular-machines.com/single_cell_sorting_products/_mmi_cellcut_-_laser_microdissection/technology_27
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