flow cytometry and SP analysis - (Jul/10/2005 )

Hi everyone,
I'm trying to isolate a side-population (SP) from tumour cells using Hoechst dye efflux and flow cytometry. The whole method seems pretty subjective, but people have published that it works and I am following their protocols. I am using 50 uM verapamil to try and inhibit Hoechst efflux (incubated 90 min, 37degreesC)

Has anyone managed to get this method to work reliably and how? I appreciate any advice because I'm not sure what to try next.

-nayo-

QUOTE (nayo @ Jul 11 2005, 04:49 AM)

Well,

The method is not exactly subjective...The sp populations is this...a populations defined by their "phenotype" in a dot plot graphic. I do the analysis in a cytometer but i didn't prepare the samples. But the thing you should do is try to isolated with a cell sorter the supposed sp populations and look for a different markers.

I could send you a protocol...but it's in catalan language....if you want...

-cafeoleman-

Thanks!

I've had some success using bone marrow cells as a control to check that the reagents are working. I did see an SP population although it was not as obvious as the published data, so I think I will repeat and try to optimise. The cells I am interested in using are from the mammary gland and I don't know if there are any other markers that I can use for sorting?

Also, from the sorting part, do you find that your samples from day-to-day are consistent? When you are defining the SP, are you using verapamil? Also, do you define the SP as the area "disappeared" when the drug is added compared to the sample with no drug?

I know someone from Barcelona, I could ask for a translation.........

Take care, Nayo

-nayo-