platinum field stimulation electrode  (Aurora Scientific)

Journal: Cell reports

Article Title: MICU2 controls mitochondrial calcium signaling and migration in neurons during development

doi: 10.1016/j.celrep.2025.116583

Figure Lengend Snippet: (A) Generation of Micu2 −/− (KO) mice. The Cre transgene under the EIIa promoter was used to target exon 3 of the floxed Micu2 (WT) gene for removal on the C57BL/6J background, as described previously. (B) Micu2 −/− mice showing Mendelian distribution. (C) Abundance of different mtCU subunits (MICU1, MICU2, MICU3, MCU, and EMRE) quantified by western blot in lysates from DIV11–15 WT and KO cortical neurons under reducing conditions normalized to the mitochondrial mass loading control, TOMM20, and WT. Significant differences ( p < 0.05) between conditions are highlighted in bold. Statistical analysis between the groups was determined using Student’s t test or Welch’s t test if criteria for equal variances are not met; n = 8 (embryos) for each group. (D) Immunodetection of MICU dimers and loading control, TOMM20, by western blot in lysates from WT and KO cortical tissue under non-reducing conditions. Representative membranes are shown. (E) [Ca 2+ ] c signals measured by Cal520 and induced by electrical field stimulation indicated on the graph in intact DIV11–15 WT and KO primary cortical neurons with Cal520 and infected with mtRCaMP; n = 4 (time pregnancies for each group). (F) [Ca 2+ ] m signals measured simultaneously with (E) as mtRCaMP fluorescence intensity and normalized to the baseline; n = 4 (time pregnancies for each group). (G) [Ca 2+ ] m signals measured simultaneously with (E) as mtRCaMP fluorescence lifetime; n = 4 (time pregnancies for each group). (H) Resting [Ca 2+ ] m in DIV11–15 WT and KO primary cortical neurons transfected with mtGEM-GECO1. Statistical analysis between the groups was determined using Mann-Whitney test; n (WT) = 10 and n (KO) = 13 (embryos). (I) Resting [Ca 2+ ] m in DIV11–15 WT and KO primary cortical neurons infected with mtRCaMP and measured as fluorescence lifetime. Statistical analysis between the groups was determined using Student’s t test; n = 4 (time pregnancies for each group). (J) Quantification of the peak for (D). (K) Quantification of the peak for (E). (L) Quantification of the peak for (F). (M) [Ca 2+ ] c signals measured by fura-2 and induced by electrical field stimulation indicated on the graph in intact DIV11–15 WT and KO primary cortical neurons with fura-2 and transfected with 4mtGCaMP6f; n = 5 (time pregnancies for each group). (N) Quantification of AUC for the first stimulation in (M). (O) Quantification of total AUC in (M). (P) [Ca 2+ ] m signals measured simultaneously with (M) as 4mtGCaMP6f fluorescence intensity; n = 5 (time pregnancies for each group). (Q) Quantification of AUC for the first stimulation in (P). (R) Quantification of total AUC in (P). Statistical analysis between the groups was determined using one-tail Welch’s t test in (J)–(L), (N), (O), (Q), and (R). p values are indicated in the graphs. Data are represented as the mean ± SEM. See also and .

Article Snippet: The cells were subjected to electrical field stimulation at 20 and 40 V/cm achieved by passing 2-ms current pulses in a biphasic mode at different frequencies using a platinum field stimulation electrode (RC-49MFSH) and High-Power Bi-Phase Stimulator 701C (Aurora Scientific) under the control of Optogenetic TTL pulse generator with corresponding software (Doric lenses).

Techniques: Western Blot, Control, Immunodetection, Infection, Fluorescence, Transfection, MANN-WHITNEY

Journal: Scientific Reports

Article Title: A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues

doi: 10.1038/s41598-021-89478-9

Figure Lengend Snippet: Differentiation of hiPSC-CM, formation of cardiac microtissues, and definitions of metrics. ( A ) Timeline shows cardiomyocyte differentiation from human-induced pluripotent stem cells (hiPSCs) in high density 2D culture. Cardiac directed differentiation was achieved with Wnt activation at day 1 and inhibition at day 3 (see “ ”). Cardiac phenotype, visually confirmed by beating cells, appeared between days 8 and 12. Cardiomyocytes differentiated from hiPSCs were used for the production of microtissues or were further purified with a lactate-based metabolic selection protocol. Microtissues self-assembled in microwells after 14–28 days of differentiation of hiPSC-CMs (i.e., without or with lactate purification) and addition of human cardiac fibroblasts (hCFs), and they were cultured in the presence of 1 Hz electrical field stimulation (estim). ( B ) Schematic of three-dimensional (3D) cardiac microtissue generation shows non-adhesive agarose gels with cylindrical recesses with hemispherical bottoms that guide self-assembly. Cardiac microtissues were cultured for 6–8 days with 1 Hz pacing. ( C ) Phase contrast image shows consistent spherical microtissue formation after 5 days of 3D culture in all 35 microwells. Scale bar, 800 μm. ( D ) Confocal image shows a representative cardiac tissue with hiPSC-CM (green) and hCF (red) stained with CellTracker dyes. Scale bar, 200 μm. ( E ) Confocal image shows a representative cardiac troponin I (red), vimentin (green), and DAPI stained cryosection (10 μm thick) of a microtissue fixed after 7 days in 3D culture. Scale bar, 50 μm. ( F ) Fluorescence image of microtissues at 3.2 × magnification was obtained during optical mapping. Typically, the action potentials (APs) from 4–9 microtissues were recorded simultaneously. ( G - I ) Schematics of the AP metrics of that were defined (with units) as: ( G ) “excitability” (%) measured from the percentage of captured APs during 10 s duration of recording with 2 s pacing cycle length, ( H ) “stimulation time delay” (ms; stim delay) between stimulation pulse and evoked AP upstroke (dF/dt max ), “rise time” (ms) of AP, “AP duration” (ms) to 30%, 50%, and 80% repolarization (APD 30 , APD 50 , APD 80 ), “APD to the maximum repolarization rate” (ms; APD MxR ) defined as time between AP upstroke and the end of rapid repolarization marked by d 2 F/dt 2 max , “APD triangulation” (ms; APD tri ) defined as APD MxR —APD 50 , and ( I ) occurrence of “early afterdepolarization” (EAD) reported as (%) of microtissues showing EADs.

Article Snippet: Microtissues were stimulated with a platinum field stimulation electrode (Supplemental Fig. , Myopacer EP field stimulator, IonOptix, Milton, MA).

Techniques: Activation Assay, Inhibition, Purification, Selection, Cell Culture, Adhesive, Staining, Fluorescence