[Cytometry] Dye cycle violet

Elliott, James james.elliott at csc.mrc.ac.uk
Sun Feb 2 09:38:20 EST 2014


In my experience almost all cells have some drug transport activity. As I remember it, this was also the conclusion of the groups who made knockouts of the various genes (mdr, mrp, abcg2 etc) - because of the redundancy of the systems, triple ko's were needed to get rid of the vast majority of activity (ie but even then perhaps not all).

I am also a bit wary of verapamil as a blocker as it seemed rather toxic when I've used it. Cyclosporin seemed to be pretty effective and tolerated better, though it is likely that no blocker will leave you with profiles as clear those you get using permeabilised cells. But hopefully good enough.

All the best

James

MRC CSC
________________________________________
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] on behalf of Cris Bare [flowmail at verizon.net]
Sent: 02 February 2014 03:43
To: Telford, William (NIH/NCI) [E]
Cc: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Dye cycle violet

I don't remember that HeLa are MDR active. I have sorted SP phenotype from MCF7 before.

One would think a cell line would be fairly homogenous for that kind of thing, but even in MCF7 SP was a small percentage.

Important to remember us that Ho42 and DCV needs time to load at 37degC in live cells. Did your user try different timepoints?

While the ethanol fixing will help give nice cycle profiles, you may lose a lot of unanchored GFP.

Three other alternatives are LDS751, Syto62, and DRAQ5. All of which will need optimization of concentration and loading time.

Cheers!
-cbb

On Jan 31, 2014, at 3:39 PM, "Telford, William (NIH/NCI) [E]" <telfordw at mail.nih.gov> wrote:

> Hi Eric...
>
> DyeCycle Violet, like Hoechst 33342, is a MDR substrate, so his cells could be pumping it out. In some cell lines this can be so strong as to completely prevent DNA binding.  One thing to try is to permeablize a sample with 70% EtOH, wash with PBS and label with an equivalent concentration of DyeCycle Violet, and analyze - if he gets good cell cycle in permeablized cells but not live, then dye efflux is likely the issue.
>
> He could also preincubate the cells with a broad spectrum efflux inhibitor like verapamil at 50 uM, and see if that improves retention.  However, this might be problematic for experimental analysis, and inhibition is not complete, so the improvement in cell cycle resolution might not be great.
>
> We use DyeCycle Violet for doing SP analysis when an UV laser is not available, and usually label between 5-20 uM final concentration.
>
> Bill
> -----Original Message-----
> From: Massicotte Éric [mailto:Eric.Massicotte at ircm.qc.ca]
> Sent: Wednesday, January 29, 2014 4:43 PM
> To: 'cytometry at lists.purdue.edu'
> Subject: [Cytometry] Dye cycle violet
>
> Hi all,
>
> I have a user which tried to stain his cell line (HeLa cells) which expresses GFP with the DyeCycle violet stain and cannot achieve to get a decent cell cycle profile. He tried different concentrations, higher and lower than the one which is suggested by the company (5uM). He just cannot resolve S and G2M from the first peak.
> Is there anyone who used that dye before on that cell type?
> Any input is welcome!
> Thanks,
>
> Eric Massicotte
> Responsable, Cytométrie en flux
> Manager, Flow cytometry
> Institut de Recherches cliniques de Montréal (IRCM)
> 110 ave. des Pins Ouest
> Montréal, Qc
> H2W 1R7
> Tél. 514-987-5627
>
>
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