Spin-down speed for HEK293 after aantibody binding?

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MattNYC
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Spin-down speed for HEK293 after aantibody binding?

Post by MattNYC » Mon Oct 19, 2009 7:17 pm

Hello,

I do antibody binding test with anti-human-IgG-PE and for the other assay with biotinylated antibody and streptavidin-apc-cy7 as the second AB. I am using 293T cells for the assay. Since the pellett is very loose I would like to increase the centrifugation speed during the washing steps. Now I use 3200 rpm (around 800g) for 4 minutes. Which speed is ok to not destroy any cells or break the antibody binding, it is analyzed by flow cytometry later?

Thanks in advance, M

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JackBean
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Post by JackBean » Mon Oct 19, 2009 10:31 pm

I think,v that e.g. 1000g for 10 min shouldn't matter ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

TaintedCherub
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Re: Spin-down speed for HEK293 after aantibody binding?

Post by TaintedCherub » Wed Oct 21, 2009 5:37 pm

I don't think you are likely to break the antibody binding by centrifugation at those sorts of speeds. I have seen reports in the literature using up to 3000 x g to pellet mammalian cells, although that seems a bit high to me. 1000 x g should be fine. If your cells are fixed you might get away with more.

If they aren't fixed and you think that you might be spinning them too fast, you could check the viability following centrifugation by resuspending the pellet, diluting a couple of ul 1 in 10 or so, mixing with an equal volume of 0.4% trypan blue solution and checking under a microscope. Viable cells should exclude the dye, dead or broken cells will turn blue.

Or you could use a live/dead stain on the flow cytometer to exclude dead cells from analysis. DAPI (if you have a UV laser) or Sytox green (if not) should fit with your other stains. This is generally good practice anyway if possible.

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