Supplementary Figure 6: Calcium imaging controls using thapsigargin.
From: Genetically targeted magnetic control of the nervous system
(a) Graph of Fluo-4 fluorescence using HEK293 cells transfected with Magneto2.0-p2A-mCherry and treated with thapsigargin over a period of 60 minutes. Arrow indicates addition of 1 μM thapsigargin to the imaging chamber after a 30 second baseline recording of calcium fluorescence. Dashed box indicates analysis window for “thapsigargin” experiments in panel b. n=114 cells analyzed from 3 independent replicates. (b) Time course showing the magnetic activation of Magneto2.0 expressing cells in the presence and absence of thapsigargin. All cells from one replicate shown per condition, n=102 cells (Magnet) and n=52 cells (Thapsigargin). In the “thapsigargin” condition, cells were pre-treated with 1μM thapsigargin and calcium imaging was initiated 30 minutes post-thapsigargin treatment during the window (dashed box) shown in panel a. (c) Quantification of maximal calcium fluorescence of HEK293 cells expressing Magneto2.0 and subjected to the above conditions using Fluo-4 calcium imaging 24 hours post-transfection. Values shown are the average maximal Fluo-4 fluorescence values per cell relative to baseline for each condition. Data points are shown as total cell averages among individual coverslips. n=114 (Thapsigargin) and n=396 (Magnet) cells analyzed from n=3 (Thapsigargin) and n=5 (Magnet) independent replicates. Welch’s two-tailed unpaired t-test, (t2.882=4.457, p=0.0395). “Magnet” data are duplicated from Figure 1. *p<0.05. Data shown as mean±SEM.
