[Cytometry] Dye cycle violet

Cappella, Paolo Elia [Nervianoms] Paolo.Cappella at nervianoms.com
Mon Feb 3 04:50:16 EST 2014


Hi Eric,

For nocodazole treatment usually we use 75ng/ml overnight with great G2M block. Of course depending to the cell lines and G2 checkpoint status you could have very different amount of M cells. For a quantification of M cells I suggest you to perform a phospho-H3 (ser 10, 28) staining. In my experience high amount of M cells after nocodazole block were reached with HT-29 colon ca cells.

Best
Paolo Cappella

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Gareth Howell
Sent: Friday, January 31, 2014 11:43 AM
To: Massicotte Éric
Cc: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Dye cycle violet

Hi Eric,

Have you tried syncing the cells in G2/M to see if it's a labelling issue or simply the cells are just super proliferative? Or maybe knock off loose cells, most of which should be G2? You can incubate cells in low concentrations of nocodazole and that arrests them in G2/M. I can't recall what concentration off the top of my head but it's in the ng/ml range overnight.

Hth

Best
Gareth
Twitter: @ghowell2812

On 31 Jan 2014, at 03:14, "Massicotte Éric" <Eric.Massicotte at ircm.qc.ca<mailto:Eric.Massicotte at ircm.qc.ca>> wrote:

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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