[Cytometry] Dye cycle violet
Cappella, Paolo Elia [Nervianoms]
Paolo.Cappella at nervianoms.com
Mon Feb 3 05:15:59 EST 2014
I completely agree with William Telford. These dyes are MRD substrates, and cells could express dirrent levels of MDR, MRP, LRP, BCRP. You should test before MDR suppressor before usage and use at not toxic concentration.
In my experience verapamil is most toxic and should be use carefully. Other inhibitors incluse a cyclosporine derivative, PSC-833 and niguldipine that in my hand seem to be less toxic in cells and in past I successfully used in LoVo/DX and MCF7/Dx cells to revert MDR activities. For a details regarding your cells there are some free databases (type on google "encyclopedia cell lines, novartis") where are indicate MDR gene expression and their levels. This can help you to used a right MDR suppressor.
All the best
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Telford, William (NIH/NCI) [E]
Sent: Saturday, February 01, 2014 12:40 AM
To: Massicotte Éric; 'cytometry at lists.purdue.edu'
Subject: Re: [Cytometry] Dye cycle violet
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.
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
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!
Responsable, Cytométrie en flux
Manager, Flow cytometry
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