Andy- I've had no problems with up to 3 days (for the blue kit, #4). I've only used the orange kit up to 24hr. I typically finish staining after 5, then fix (0.5% EM grade formaldehyde) & wash off the fix the next a.m., {resuspend in PBS/0.5% BSA/0.02%azide} Then I run anywhere from that day (24hr) to 3 days. (I have not actually tested it longer than 3 days, that's just my time frame for getting my samples run). I have used Live/dead for intracell, but those are generally run within 24hr ; so I have no idea how those samples hold up under storage. bunny On Oct 25, 2005, at 5:17 AM, A Herman, Cellular & Molecular Medicine wrote: > Dear Bunny, > How long do you leave the fixed cells with the Live/Dead stain > before you analyse them on a cytometer? > > Molecular Probes tech support said "ideally you would run them > immediately but should be fine the next day when kept at 4C, > degradation would most likely occur after a couple of days". Has that > been your experience? > > Also, have you used these dyes with cells that are then permeabilised > for intracellular staining? > > Many thanks, > Andy > > --On Monday, October 24, 2005 2:33 -0400 bunny <bunny@cotleur.com> > wrote: > >> Paolo- >> I've been using Molecular Probes Live/dead kits for about 2yrs now >> with >> great success. Although I use #4 (runs off the UV) I've also tried >> #2 >> which works well off 488nm. You incubate the cells for 30min, then >> are >> able to fix to analyze later. >> >> Good Luck! >> bunny >> >> >> Bunny Cotleur, M.S. >> Sr Research Technologist, Dept of Neurosciences >> Scientific Consultant, Flow Cytometry Core >> Cleveland Clinic Foundation >> Lerner Research Institute, NC30 >> 9500 Euclid Avenue >> Cleveland, OH 44195 >> Lab: (216)444-1164 >> ********************************************* >> Caminante, no hay camino. Se hace camino al andar. >> >> On Oct 21, 2005, at 8:41 AM, paolo wrote: >> >> Dear Fellow Flowers, >> >> I work with HIV infected cultures and I need to determine apoptotic >> vs >> live vs dead cells. I use an Coulter XL with a single laser (488nm) >> and I >> have to fix cells with 1% PFA as they are loaded with HIV. If I use >> PI or >> 7AAD all cells will turn dead after fixation. Can anyone suggest >> alternatives to these dyes? I already tried EMA, but perhaps there >> something else out there that does not require the extra step with a >> light source... >> >> Thanks in advance for all your help. >> -- >> >> ============================ >> Aut viam inveniam aut faciam >> ============================ >> Paolo Piazza, PhD >> OFFICE: 604 Parran Hall >> tel (412) 383 - 9590 >> LABORATORY: 529 Parran Hall >> tel: (412) 648 - 2940 >> Infectious Diseases and Microbiology >> GSPH ? University of Pittsburgh >> 130 Desoto Street >> 15261 Pittsburgh PA >> fax (412) 624 ? 4953 >> e-mail paolo@pitt.edu >> >> >> > > > > ---------------------- > Andrew Herman, Ph.D. > University of Bristol > Department of Cellular & Molecular Medicine > Flow Cytometry Facility > School of Medical Sciences > University Walk > Bristol BS8 1TD > UK > > > A.Herman@bristol.ac.uk >Received on Wed Oct 26 15:58:00 2005
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