B cells were loaded for 60 min at 37°C with Indo-1 acetoxymethyl (final concentration, 1 µM; Molecular Probes) in RPMI containing 10% FCS. Baseline measurements were acquired for 30 s, and then cells were stimulated with 10 µg/ml F(ab′)2 goat-anti-mouse IgM (Jackson ImmunoResearch) or 2 µg/ml TG2 for 2.5 min. Mean relative [Ca2+]i was monitored over time using an LSR II flow cytometer (BD) with analysis using FlowJo software (Tree Star).
Indo 1 acetoxymethyl
Manufactured by Thermo Fisher Scientific
Indo-1 acetoxymethyl is a fluorescent calcium indicator that can be used to measure intracellular calcium levels in live cells. It is a cell-permeant form of the calcium-sensitive dye Indo-1.
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Lab products found in correlation
2 protocols using indo 1 acetoxymethyl
1
Intracellular Ca2+ Measurement in B Cells
2
Measuring Calcium Flux in PU.1 SpiB DKO B Cells
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Naive resting B cells were isolated as described above and cultured for 48 h in CD40 L + IL-4, to ensure complete deletion of the Spi1 allele in PU.1 SpiB DKO B cells. Cells were then washed extensively and rested for one hour before being resuspended in loading buffer (KDS-BSS buffer containing 0.1% glucose and 0.1% FCS) containing Indo-1 acetoxymethyl (Molecular Probes). Following an incubation for 40 min at 37 °C, loaded cells were washed extensively and then resuspended in the loading buffer indicated above. Calcium flux was measured by flow cytometry. Baseline fluorescence was established for 30 s before anti-IgM (10 μg/ml; Jackson Immunoresearch) was added and the subsequent calcium flux followed for the indicated times.
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