We did a lot of work of flow methodology for glutathione a few years ago, and published a detailed evaluation of the subject in Cytometry (1994;15:349-58). Our conclusion at that time was that the only effective stains are monobromobimane and monochlorobimane. The latter is fairly non-reactive, but is conjugated to glutathione via the action of some glutathione S-transferases. This makes it more specific than monobromobimane (which is chemically more reactive), but this specificity is at the expense of underestimating GSH levels. Therefore we recommended monobromobimane. Horan et al. published an evaluation of our protocol, backed up up by HPLC correlates, and confirmed that this gives a reliable estimate of cellular glutathione. This area is a bit tricky because the act of labeling for GSH is toxic, and has major consequences for cellular redox systems. Furthermore, prolonged staining times and concentrations result in background staining of protein -SH groups. Also, the act of detaching cells from monolayers can deplete GSH. So far as I am aware, our experience remains current state of the art, although I would be interested to hear if anybody has other ideas. A problem with the bimanes is that they require UV excitation. David Hedley Ontario Cancer Institute Toronto > -----Original Message----- > From: Scott Tighe [SMTP:stighe@zoo.uvm.edu] > Sent: Friday, March 02, 2001 4:45 PM > To: Cytometry Mailing List > Subject: Glutathione detection > > > Dear Flowers > > We need to detect Glutathione in rat tumor cells and understand that > only several dyes exist from mostly Molecular Probes. I would like to > hear from anyone that has tried to do this on their cell line using > flow. I have read that data collected using flow can be somewhat > ambiguous. Have you been successful? Which dyes can be used? Which work > best? What are the drawbacks? > > Scott Tighe > Vermont Cancer Center > Flow Cytometry Core Lab > 802.656.1333
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