Flow cytometric analysis of murine splenic B lymphocyte cytosolic free calcium response to anti-IgM and anti-IgD.
The accurate measurement of ionized intracellular calcium [Ca++]i in single cells by flow cytometry with the use of a new fluorescent calcium chelator, indo-1, is described. We have developed a dependable in situ calibration technique that indicates a resting [Ca++]i in lymphocytes of 100 nM. The enhanced fluorescence of this probe permits its use at sufficiently low cytoplasmic concentrations that buffering of [Ca++]i transients does not occur. The [Ca++]i response of small resting B lymphocytes to crosslinking of surface antigen receptors by anti-immunoglobulin is heterogeneous. With maximal stimulus, the peak [Ca++]i response occurs in 10 to 20 seconds with most cells reaching levels greater than/1 microM. Mean [Ca++]i falls to between 300 and 800 nM by 100 seconds where it remains for more than 10 min. Anti-delta is a more potent stimulus of increased [Ca++]i than anti-mu in terms of both [Ca++]i level and fraction of B cells responding. Whether this is due to the greater density of surface IgD than IgM, a difference in signal transduction efficiency, or both, is not yet known. Surface immunoglobulin receptors are present in great excess. Less than 3% of surface immunoglobulin is crosslinked at the peak of the [Ca++]i response.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D007072:Immunoglobulin D / Q000276:immunology*; D007132:Immunoglobulin Isotypes / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology*; D007211:Indoles; D051379:Mice; D008807:Mice, Inbred BALB C; D011947:Receptors, Antigen, B-Cell / Q000276:immunology; D011971:Receptors, Immunologic / Q000378:metabolism; D013154:Spleen / Q000166:cytology
D000888:Antibodies, Anti-Idiotypic; D005456:Fluorescent Dyes; D007072:Immunoglobulin D; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D007211:Indoles; D011947:Receptors, Antigen, B-Cell; D011971:Receptors, Immunologic; C039951:anti-IgD; C044203:anti-IgM; C048960:indo-1; D002118:Calcium