Labtek chambers

HeLa cells were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 60 µg/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator (Heraeus, Thermo Fisher Scientific Inc., Germany) at 37 °C and 5% CO₂. Cells were seeded in eight-well LabTek^TM chambers (Nunc, Langenselbold, Germany) and allowed to adhere overnight in a humidified incubator at 37 °C and 5% CO₂ before they were washed twice with PBS.
Human MSCs were obtained from bone marrow aspirates or explanted hip bones [50 (link)] and cultured in alpha minimal essential medium (R-MEM, Cambrex, East Rutherford, NJ) supplemented with 20% fetal calf serum (FCS), 100 U penicillin, 100 mg/mL streptomycin, and 1 mM pyruvate (Sigma-Aldrich) in a humidified incubator (Heraeus, Hanau, Germany) at 37 °C. For confocal imaging, cells were seeded at a density of ≈10,000 cells/cm².

Unless indicated all images were acquired on a Leica SP8 Lighting Confocal microscope. For live imaging experiments, cells were grown in Lab-Tek^TM chambers (Nunc) with a borosilicate glass bottom. Media was replaced 30 min before imaging and cells were imaged using a Leica SP8 with a 63x glycerol immersion objective lens, 1.3 NA (Leica) in a temperature (37 °C), and atmosphere (5% CO2) controlled environment. Depending on the experiments, single optical section or a Z-stack of 3–5 steps with a step size ranging from 250 nm to 340 nm were acquired. Acquisition was done using the Las X software (Leica) and a deconvolution was performed on every image with the lightning module (Leica) using adaptative parameters. For lysosomes-mitochondria contacts and mitochondrial division experiments; a video of 1 or 2 min with 1 frame every 5 s was acquired. When the presence of the endoplasmic reticulum (ER) at contacts between lysosomes and mitochondria was investigated, or when contacts between lysosomes and the ER were investigated, a 15–30 s video was acquired. Cells imaged were of similar low intensity in order to be able to compare cells with similar levels of overexpression.