I have an investigator who is trying to perform the calibration curve for intracellular [Ca++] following essentially the method in Current Protocols in Cytometry except that they purchased the buffers from Molecular Probes. While I have done a lot of indo-1 Ca++ I have never myself nor had any one do this calibration (probably should have by now but I haven't). The cells (mouse lymphocytes) look terrible (i.e they all look dead) after they are placed in the calibration buffers and following treatment with the agents to open up the Ca++ channels, etc. FSC is much decreased and the indo-1 loading levels have dropped to the point that I have to turn up the PMT voltages a lot to get the indo-1 405 and 485 signals back to where loaded cells in regular media are. Also, although we can see the ratio increasing as we raise the [Ca++] it doesn't appear like that in Current Protocols - the increases are too small. This strikes me as not the way to have to do the calibration. Has anybody done this calibration (especially with the Mol. Probes buffers)? Is what I'm seeing normal or do I have to try to figure out what the heck this investigator is doing? Thanks Larry Larry W. Arnold, Ph.D. Associate Professor Director, Flow Cytometry Facility Department of Microbiology and Immunology Lineberger Comprehensive Cancer Center CB# 7290 University of North Carolina at Chapel Hill Chapel Hill, NC 27599 Phone: 919-966-1530 FAX: 919-962-8103
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