Hi everyone:) i am using magnetic beads with specific antibodies to seperate macrophages. After the macrophages have been separated by positive selection, How do i seperate the macrophages from the magnetic beads?
If you are using Dynabeads please see the attached protocol from Invitrogen.https://www.invitrogen.com/content/sfs/manuals/114.41D%20Dynabeads%20Mouse%20pan%20B%20(rev006).pdf
Look at section 2.3
Hi again:) well i am using magnetic particles made by a Russian firm, but the principle is actually the same. I have gone thru the protocol, but it does not say anything how to separate the macrophages from the magnetic beads. Any ideas?
From What I anderstand in section 2.3.5
5. For positive isolation, discard the supernatant
and wash the bead-bound cells 3 times by resuspending
in PBS to the original sample volume,
and separate using a magnet.
After the reaction with the beads and using the magnet the beads that reqacts with the magnet will have the Macrophages coupled .
So I guess afte taking off the Magnet you can spin the tube in a low G as the beads are much more heavy from the cells you can take the sup after spining the beads down.
I guess that the sup will have the cells that you are looking for .
Hey thanks Guy, i will try that.
I am a support scientist at Invitrogen Dynal. We have many different Dynabeads and a few different methods of detachment of the beads from the cells. In general, unless you are using a bead to which the antibody is attached via a molecule which can be competed out or digested, you will not be able to detach the cells from the beads.
For molecular studies, detaching the beads is not necessary, as you can simply lyse the cells while still attached to the beads. Then, once the cells are lysed you can place the lysate on a magnet and transfer the bead-free lysate to a clean tube.
If you wish to do cellular studies, e.g. FACS or stimulation etc, it is reccomended to detach the cells. I would reccomend Dynabeads FlowComp Flexi together with your antibody. After isolation you can detach the cells from the beads by adding the detach solution which comes with this kit.
More info here: http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=11061D&CID=Search-11061d
I hope this is helpful