Calcium buffering capacity of neuronal cell cytosol measured by flash photolysis of calcium buffer NP-EGTA

N1E-115 mouse neuroblastoma cells were injected with a calcium buffer/indicator solution to allow both ratiometric measurement of free calcium concentration and the release of calcium ions upon UV flash. The solution contained sulforhodamine, a marker dye used to estimate the volume injected; fluo-3, a calcium indicator, and NP-EGTA, a high affinity calcium-selective buffer that is converted by UV hash to products with negligible calcium affinity. The calcium increase recorded upon UV irradiation (Delta[Ca(2+)](i)) was small for small injection volumes, increased with larger injection volumes, but approached a plateau at the largest injection volumes. From this relation we estimate the buffering capacity of the cytosol as 1700 ions bound perion free. (C) 1998 Academic Press.