I want to thank all those that responded to my query about macrophages fluxing calcium in response to pressure changes about a month ago. I got many answers. Most respondants suggested that the machine is dirty. We know that not to be true, and the fact that the flux only occurs after pulling the macrophages off the machine adn putting it back on suggests that it is physical stimulation. When we used the standard Vantage collection apparatus, we decreased the calcium flux, but did not abrogate it. We tested this by putting the Vantage pressure tubing through the time zero apparatus, and that improved but did not abrogate the problem. We also improved the calcium flux by turning down the sample pressure, but could not abrogate the problem. We actually have realized that the problem began when we replaced the top part of the housing of the Time Zero apparatus because a small crack had gotten to large. In retrospect this crack was probably dampening the pressure changes and allowed us to get good data. We are going to try some pressure sensitive valves in line to see if we can "reproduce" the crack. Dr. Tarnok suggested the time-window method published in Cytometry, and we may go to that. We are also going to try it on a FACScan with Fura-red and fluo-3 because the pressure of the fluidics is so much lower. Thank you for your suggestions. 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
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:30 EST