Figure 2.

Magnetic activation of Magneto2.0 is thermally mediated. (a–j) Distribution of intracellular calcium levels over time based on calcium-sensitive fluorescence imaging (ΔF/F₀) is shown. In the absence of stimulation (a–e), distribution broadens over time but remains centered near zero. Under periodic magnetic stimulation (275 mT, 0.08 Hz, beginning at t = 30 s), a small percentage of cells (seen in red tail of the distribution) show an increase in calcium-sensitive fluorescence for Magneto2.0 (f) and (−)Mech (g), shifting the mean of the distribution. This is not the case for (−)Therm (h) and Magneto2.0 exposed to a sham magnet (i) or to a constant magnetic field (j). (k–o) Histograms taken from the data in (a)–(j) show the distribution of fluorescence values at t = 270 s with no magnetic stimulation (black) and with magnetic stimulation (red) (bin size 0.02 ΔF/F₀). These histograms correspond to the white lines in (a)–(j). Vertical red and black lines represent the mean value of these distributions with and without magnetic stimulation, respectively. Error bars show the mean ± SE for each histogram. (p–t) Plotting the mean value of ΔF/F₀ over time shows statistically significant responses for Magneto2.0 (p) and (−)Mech (q) in response to magnetic stimulation but not for (−)Therm (r) or Magneto2.0 exposed to a sham magnet (s) or to a constant magnetic field (t). ΔF/F₀ values from each cell culture are averaged using a 20 s sliding window. The resulting average and mean ± SE (calculated using n = number of independent cell cultures) are shown as a solid line and a shaded region, respectively. The periodic artifacts most visible in (q) and (r) result from a small reversible movement of the microscope stage that sometime accompanied magnet movement. Equal numbers of “No Stimulation” and “Magnetic Stimulation” experiments were performed for each condition. (u–y) Each dot represents the area under the curve of the calcium activity calculated for a given cell culture. The average area under the curve for all cell cultures is shown as a horizonal bar with error bars indicating the corresponding mean ± SE, calculated using n = number of independent cell cultures. The significance of the increase in calcium activity was assessed using the left-tailed Wilcoxon test. This nonparametric test is used because the increased cell activity is not necessarily normally distributed. ^∗p < 0.1; ^∗∗p < 0.05; ^∗∗∗p < 0.01. The total number of cells measured from separate cell cultures are (indicated as total number of cell/number of separate cell cultures) Magneto2.0, n = 1573/14 (no stimulation), n = 1510/14 (magnetic stimulation); (−)Mech, n = 1290/11 (no stimulation), n = 1587/11 (magnetic stimulation); (−)Therm, n = 1724/10 (no stimulation), n = 1073/10 (magnetic stimulation); Sham Stimulation, n = 2759/19 (no stimulation), n = 2970/19 (sham stimulation); Constant Magnet, n = 1536/12 (no stimulation), n = 1692/12 (magnetic stimulation). Note that prior studies of magnetogenetic channels often calculate mean ± SE with n representing the number of cells. In that case, the error bars would be significantly smaller than the error bars shown here.