Mayumi, The NIH doesn't have a cytometer with a UV source to excite indo-1?! In my opinion, indo-1 would be a better way to go. However, you may want to check out the dyes available from Molecular Probes <http://www.probes.com/>. In particular, the combination of fluo-3 and Fura Red allows for a more sensitive ratiometric measurement (if your software can perform the ratio, like FlowJo, since the FACScan cannot ratio in hardware) as described in: Novak EJ, Rabinovitch PS: Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios. Cytometry 17(2): 135-41, 1994. You may also be interested in paring down this 4-color, non-ratiometric assay to 3-colors: Greimers R, Trebak M, Moutschen M, Jacobs N, Boniver J: Improved four-color flow cytometry method using fluo-3 and triple immunofluorescence for analysis of intracellular calcium ion ([Ca2+]i) fluxes among mouse lymph node B- and T-lymphocyte subsets. Cytometry 23(3): 205-17, 1996. Also, Molecular Probes indicates in their catalog that the Oregon Green 488 BAPTA dyes should be superior to fluo-3; unfortunately, I was not able to find, using NCBI PubMed <http://www.ncbi.nlm.nih.gov/PubMed/>, any references where this dye was used for flow cytometry. Finally, a recent review of Ca2+ indicators can be found in: Simpson AW: Fluorescent measurement of [Ca2+]c. Methods Mol Biol 114:3-30, 1999. Good luck, Eric >Dear Flow people, >I am trying to measure intracellular Ca using a regular FACScan. I would >greatly appreciate your opinions about dyes excitable at 488 nm (not Indo-1 >or Fura-2.) Especially, if you have worked with fresh mouse splenocytes/LN >cells, I would love to hear from you. (Any tips for loading cells with dye, >compatibility with surface staining etc.) >Thank you >Mayumi >Mayumi Naramura, MD >NIH/NIAID >Laboratory of Immunology >Twinbrook II, Room 125 >12441 Parklawn Drive >Rockville, Maryland 20852 >Phone: 301-402-4595 >FAX: 301-594-2522 >E-mail: mnaramura@niaid.nih.gov /\/\/\_ Eric Van Buren, aa9080@wayne.edu \ \ \ Karmanos Cancer Institute and Immunology & Microbiology \_^_/ Wayne State University, Detroit, Michigan, USA
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:21 EST