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. 2019 Sep 3;9:12692. doi: 10.1038/s41598-019-49070-8

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Preliminary comparisons of 293-F cells labelled with GCaMP6s or with fluo-4. (a–c) Histogram of log-transformed fluorescence intensity by flow cytometry for (a) unlabelled cells, (b) transient expression of pGP-CMV-GCaMP6s, and (c) cells labelled with fluo-4. Cells were treated with buffer (red) or with 10 µM ionomycin (blue). (d) Schematic representation of the GCaMP6s-P2A-Bsr construct. During translation, the P2A linker fails to form a peptide bond between glycine and proline. GCaMP6s and Bsr emerge as separate proteins, with fragments of the P2A linker at the C- and N-termini as shown. (e,f) Time-course of fluorescent signal for stable 293F.GluA1o-γ4 during stimulation with 15 µM glutamate. (e) Transiently-transfected GCaMP6s-2A-blast. (f) Labelled with fluo-4. Black lines indicate n = 40 negative control wells (0.5% DMSO only); blue lines indicate n = 40 control wells pre-incubated with a PAM (10 µM LY-395153); red lines indicate n = 40 positive control wells pre-incubated with an inhibitor (50 µM CP-465022). Vertical grey lines indicate the position of the measurement window.