Hellow everyone!
I am using eBiosciense magnetic beads kit for MACS separation of murine CD11b+ cells (= monocytes and granulocytes).
It consists of Biotin-labelled anti-CD11b Ab; and a secondary agent: streptavidin crosslinked to magnetic particle.
But when afterwards running flow cytometry, with fluorochrome-labelled anti-CD11b Ab, I'm getting quite poor CD11b+ enrichment yield.
Does it mean that MACS did not work properly?
Or, maybe, that Biotin-linked anti-CD11b Ab which is used for MACS, which has already stuck to antigen, obstructs fluorochrome-linked Ab and prevents it from contact with Ag?
After MACS separation, any ways exist to wash off MACS reagents off the cells surface?
Maksym,
There are two possible routes here - using a conjugated CD11b ab specific to a different epitope as suggested by several others. Or, you can make use of the CD11b-biotin ab already bound to the cells and use a streptavidin secondary conjugated to a fluorochrome. You need to use it at a concentration about 10x your normal concentration in order to drive off the streptavidin-bead complex and move the 'steady-state' towards binding the fluorochrome SAv. Remember - the streptavidin-biotin complex is not covalent and a second streptavidin ab will have the same affinity as the first SAv ab. We have used this method quite effectively in our lab many times.
Hi there,
You yourself have proosed a few possible reasons. First, your anti-CD11b antibody (and the microbeads) might successfully obscure the access of fluorescently-tagged Ab or at least to thhe epitope already 'occupied' by Ab-bead complex. The manufacturer recomends that after positive selection, the purity of selected cells can be verified by staining with Anti
Mouse CD11b, clone M1/7 . Did you use that one?
As you do not write what material you isolate your monocytes from, it is hard to say what else might be in your MACS-separated suspension. If it were - say - mouse spleen - you could expect plenty B and T lymphoid cells if your separation were poor; so, you could stain your pr-and postseparation cell suspensions for say murine CD3 and B220 and you should know if your lymphocid contamination is acceptable or not. Also, if you isolating tissue macrophages I would suggest similar approach.
I do not know af a reliable protocol to remove ebioscience beads from the cell surface and mostly it seems not to be necessary, as they are really small compared to the cell size....
I had a similar problem with the CD11b magnetic beads from Miltenyi. I called the company and they said this shouldn't be a problem, since the beads are so small, but they also said there is no way that they know of to get the beads off. I think Jacek is right, that you should try to use an antibody for flow that binds to a different epitope than the beads do. Good luck!
Thanks Jacek and Jessica for meaningful answers!
Jacek - yes, it's splenocytes, bone marrow cells, and erythrocyte-depleted blood.
Yes - Ab clone (for FCM and, I guess, for MACS also) is M1/70 indeed.
Jessica - nice to know that someone had already raised this question before; and strange to hear that even at Miltenyi they don't know how to remove the beads...
If your experimental epitope is different than that interacting with bead than no problem. In general Magnetic sorting beads bind to other site than active epitope so that downstream experiments will not be hindered.
Maksym,
There are two possible routes here - using a conjugated CD11b ab specific to a different epitope as suggested by several others. Or, you can make use of the CD11b-biotin ab already bound to the cells and use a streptavidin secondary conjugated to a fluorochrome. You need to use it at a concentration about 10x your normal concentration in order to drive off the streptavidin-bead complex and move the 'steady-state' towards binding the fluorochrome SAv. Remember - the streptavidin-biotin complex is not covalent and a second streptavidin ab will have the same affinity as the first SAv ab. We have used this method quite effectively in our lab many times.
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