Charles, the simplest assay that comes to mind is propidium iodide exclusion. Any cell with a damaged membrane will allow PI to stain the DNA content, whereas viable cells will normally not allow PI to enter and stain. The number of PI +ve cells will give you a good idea of the number of dead/dying cells. Eric P Miller Edinburgh Medical Oncology Unit Legal bit: ************************************** The information contained in this message is confidential and may also be privileged. The disclosure, copying or alteration of this message is strictly prohibited. This message is intended for the addressee/s named above. If you are not the addressee/s (or responsible for the delivery of the message to the addressee) please notify the originator immediately by return message and destroy the original message. The originator does not guarantee the security of this message and will not be responsible for any damages arising from any alteration of this message by a third party. ************************************** On Mon, 30 Oct 2000, charles wymond symes wrote: > > Hello everyone, > > I am a lab technician with no flow cytometry experience whatsoever. I > am hoping to develop a simple bioassay to quantitate glutamate toxicity > in differentiated P19 cells. > Differentiated P19 cells express functional NMDA receptors. The ligand > for this receptor is glutamate. however at high levels glutamate acts > as a neurotoxin and the resulting influx of calcium through the > receptor channel is fatal to the cell. The assay I am hoping to develop > will quantitatively examine the protective effects of selective NMDA > receptor channel blockers. I plan to use facscan analysis to determine > the percentage of dead cells within populations of cells that have or > have not been treated with the blockers prior to addition of a fatal > dose of glutamate. I could use TUNEL staining or some other marker of > cell death but this is quite a long-winded affair due to the > involvement of several antibody incubation steps. Is there a quicker > alternative. > > Thanks for taking the time to read this. I hope some of you have some > ideas, > > Wymond Symes > > > ---------------------- > > Charles Wymond Symes > CNS Gene Therapy Centre > Dept. Molecular Medicine > University of Auckland > Private Bag 92019 > Auckland > ph 3737599 xtn 4482 > fax 3737492 > >
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