Calcein am

Mitochondrial-specific ATP was assessed after permeabilizing the cell membranes (Zoeteweij 1994 (link)) (supplementary Fig. 2F). After the desired exposure period Hanks’ buffer (Thermo Fisher 14175053) was supplemented with 5 mM HEPES, 250 mM sucrose (Thermo fisher s8600/63), 25 mM TRIS, 3 mM EGTA (Sigma-Aldrich 4378), 5 mM MgCl₂ (Sigma-Aldrich 8266), 5 mM succinate (Sigma-Aldrich S2378) and 5 mM glutamate (Sigma-Aldrich G5889) (37 °C, pH 7.3). 150 µM digitonin (Sigma-Aldrich D5628) was added to permeabilize the cell membranes. After 30–45 s exposure, the buffer was replaced by PBS and the ATPlite 1step Luminescence Assay was performed as described above. The membrane permeabilization protocol was validated using confocal imaging. For this, cells were co-stained with Hoechst and 0.5 µM Rho123 and/or 0.05 µM Calcein–AM (VWR, 734–1434), to assess mitochondrial integrity (digitonin exposure should not affect the Rho123 intensity; Suppl. Figure 1F and G) and loss of cell membrane integrity (digitonin exposure should result in the loss of Calcein-AM signal; Suppl. Figure 1F, H and I). The Hoechst (408 nm) and Rho123/Calcein-AM (488 nm) signal intensity was monitored live every 10 s just before and after addition of digitonin using a 20 × objective on a Nikon TiE2000 with perfect focus system, automated-stage, and controlled temp/CO₂ incubator (Nikon, Amsterdam, The Netherlands).