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. 2021 May 21;11:10714. doi: 10.1038/s41598-021-88906-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Quantification of calcium release events in response to glucose and NAADP-AM in low EGTA (100 μM). (a–c) Representative TIRF traces of β-cells stimulated with: (a) 100 nM NAADP-AM after preincubation with thapsigargin, (b) 16.5 mM glucose after preincubation with thapsigargin, (c) 6 mM glucose without thapsigargin preincubation. (d) Epifluorescence recording (all other parameters equal) using 6 mM glucose without thapsigargin preincubation. Maximum intensity change of subsequent frames after normalising to baseline plotted against time. Insets show magnified events for each of the three experimental stimuli, chosen to illustrate the variation in event size. (e–g) Percentage of frames showing elementary events (defined as events of an amplitude more than 2 standard deviations above baseline mean) before (baseline) and after stimulus addition. (e) NAADP-AM; n = 20 (8 experiments, 4 Animals), t(19) = 7.90, p < 0.01, 90% response rate. (f) 16.5 mM glucose; n = 14 (6 experiments, 4 Animals), t(13) = 7.44, p < 0.01, 100% response rate. (g) 6 mM glucose; n = 18 (6 experiments, 6 Animals), t(17) = 2.61, p < 0.01, 50% response rate. * denotes significance; paired samples, one-tailed t-tests.