Improved method for measuring intracellular Ca++ with fluo-3.
The accuracy of flow cytometric measurement of intracellular calcium with fluo-3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo-3 and SNARF-1. When SNARF-1 fluorescence is collected at approximately 600 nm, its intensity does not change upon cell activation. Furthermore, fluo-3 and SNARF-1 fluorescence signals exhibit a linear relationship. The ratio of fluo-3 to SNARF-1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo-3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i.
Rijkers
G T
GT
Department of Immunology, University Hospital for Children and Youth, Het Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands.
Justement
L B
LB
Griffioen
A W
AW
Cambier
J C
JC
D000814:Aniline Compounds; D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry; D001578:Benzopyrans; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D014966:Xanthenes
D000814:Aniline Compounds; D001578:Benzopyrans; D005456:Fluorescent Dyes; C063917:SNARF dye; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium