Hello and Happy Holidays! I have some questions about measuring pHi using SNARF-1. Throughout the literature different groups have somewhat different methods. I would appreciate any input in this matter. 1. Is there any advantage to incubate the dye-loaded cells in culture media (bicarb containing) as opposed to incubating them in buffer? Is it necessary to wash the cells after dye incubation? 2. Some groups place the dye loaded cells in ice before analysis. Is there any benefit to this? 3. In order to get a calibration curve, the cells are placed in buffers at different pH and before analysis Nigericin is added to equilibrate pHi and pHe. Should the dye-loaded cells be incubated in these buffers? 4. The flow cytometer that we have is a single laser (Argon 488 nm). The only channel that I have available for measurements in addition to pHi would detect emission around 525 nm. We are unable to sort cells. I am interested in monitoring the apoptotic population and relate it to the pHi. I was thinking about using Annexin-V (FITC). However, this is usually used along with PI in order to differentiate the different populations. Is it correct to assume that cells that do not hold SNARF-1 are necrotic? Would Annexin-V and this assumption be sufficient to establish the different populations? Is there a less expensive alternative to Annexin-V? Thank you very much in advance, Hope to hear from you soon :) Vivian Vivian de Zengotita Northwestern University Chemical Engineering Dept. Evanston, IL (847) 491-3519 dezengot@nwu.edu
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