David McFarland asks: > Is it feasible to do calcium flux experiments on a Calibur or FACStar+ > without a > lot of extra hardware? We're thinking of using Fluo4 to start and then a > Fluo4/Fura Red combination. We don't have a way to regulate the > temperature of > the sheath or the sample. Are we just out of luck? I know that Cytek > makes a > module for speeding delivery of the sample to the interrogation point, but > can > we get along without it? (That's a hefty price to pay for hardware that's > only > going to be used by a couple of investigators.) And will it work on the > Calibur? I would appreciate any advice. > > David McFarland > Howard Hughes Medical Institute > Flow Cytometry Facility > Vanderbilt University Medical Center > David, there are a few variables in your experimental system which are unstated. Are you looking at a heterogeneous population of cells? If so, ratiometric analysis is critical, since different cells (T cells, B cells, monocytes) load differently. I have no real experience with Fluo/Fura red combinations, though I gather from comments of others suggest that it is not as clean as indo-1. If you have access to a UV laser, I would use indo-1, especially if you are looking at a heterogeneous population of cells. Regarding the Cytek module, this depends on the stimulus and the cells you are looking at. Most certainly you do not need the cytek module for a response that has a delayed onset and is prolongued, such as stimulation through CD3. For Gi protein coupled responses, that are lower in amplitude, of rapid onset, and short duration, I have found the Cytek module to be critical. Cytek does make a module that will work on the Calibur. Regarding temperature, this response is temperature sensitive. Lymphocytes I always run at 37 degrees. Macrophages we have actually done at room temperature to try to decrease their inherent responses to just being looked at. So this depends on your experiment once again. ron > Ronald L. Rabin, M.D. > Clinical Associate > Cytokine Biology Unit, Laboratory of Clinical Investigation > National Institute of Allergy and Infectious Diseases > National Institutes of Health > Bldg. 10/Rm. 11N228 > 10 Center Drive MSC 1888 > Bethesda, MD 20892-1888 > Phone: (301) 402-4910 > FAX: (301) 402-0627
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