I need some help regarding a problem that was small initially, but is now insurmountable. We have been following the calcium response to chemokines in macrophages using a FACSVantage equipped with a Time Zero module. The macrophages have always been sensitive to physical stimulation--we do not use a stir bar and try to run at low sample pressures. Occasional donors would have a calcium flux, small but definite, with injection of buffer alone, without ligand. Lately, the flux in response to injection of buffer has been huge, making it impossible to accumulate any new data. Putting the cells on the cytometer, taking them off, and then putting them back on even causes a calcium flux, so the cells are responsive to changes in pressure, agitation or perturbation, but only after initially being placed on the cytometer. In the words of Dave Barry--I am not making this up. EGTA does not suppress the flux in response to buffer injection, so this is a release from intracellular stores. The cells seem to behave on a fluorimeter, which must have a stirring apparatus, so I think the positive pressure of sample collection is an important component of the problem. We have tried lowering the sheath pressure, and that does decrease the problem, but not enough. We have tried different temperatures (room temp, 30, 37) and different loading parameters. We use HBSS with Ca and Mg, 10mM HEPES, 1% FBS as our buffer for cell suspension, but have tried getting rid of the serum, using only PBS--no help. We have changed lots of media and serum in which the mphages are grown (RPMI, 10%FBS and human serum, and GMCSF). All to no avail. To our knowledge, nothing about the machine has changed. Anyone have a clue? We are at a point of finishing studies to write them up, and now we have hit this brick wall. Thanks. 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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