Hela cell

HeLa cells (ATCC, Manassas, VA), HeLa cells stably transfected with GFP-tagged beta 1,4-galactosyltransferase (GalT) (gift from Dr. Jack Rohrer, Zurich University of Applied Sciences, Wädenswil, Switzerland), and HeLa cells stably transfected with GFP-tagged tubulin (gift from Dr. Ryoma Ohi, University of Michigan, Ann Arbor, MI) were cultured in MEM media (Thermo Scientific, Waltham, MA) supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% sodium bicarbonate, 1% PenStrep, and 0.2% NEAA. Cells were grown at 37 °C and 5% CO₂. Cell transfection was performed with TransIT-LT1 transfection reagent (Mirus Bio, Madison, WI) according to the manufacturer’s instructions.

Porcine kidney LLC-PK and human cervical cancer HeLa cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were maintained in Eagle’s minimal essential medium (EMEM). Human intestinal Caco-2 cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM). Monkey kidney MA104 cells (ATCC) were grown in alpha minimal essential medium (α-MEM). Each culture medium was supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The PSaV Cowden strain was generated from the full-length infectious clone pCV4A and was propagated in LLC-PK cells in the presence of 200 μM GCDCA [23 (link)]. CVB3 Nancy strain (ATCC) was propagated in HeLa cells [31 ] and then used for the subsequent studies with Caco-2 cells [31 ]. The human rotavirus strain Wa (G1P [8 (link)]) (ATCC) was preactivated with 10 μg/mL crystalized trypsin and propagated in MA104 cells as previously described [32 (link)].

For dynamical studies of mitochondria:
HeLa cells obtained from American Type Culture Collection (ATCC) were cultured and stained with mitotracker as described in our previously reported study^{43 (link)}. To mount the sample vertically to be adaptable in our home-built (M $\lambda$ -sMx-SPIM) microscopy system, the cell was cultured in a glass bottom dish (Cell Vis, D35-20-1.5-N). After 18–24 h of plating and staining with MitoTracker^TM Red CMXRos, the bottom was filled with a small amount of DMEM solution and then, sealed with a cover slip using clear nail polish. The samples were immediately used for imaging. This is to note that the glass bottom was kept vertically by using a custom-made mounting base on the sample mounting stage (mentioned above).
For the multispectral imaging:
The nuclei of HeLa cells (obtained from American Type Culture Collection (ATCC)) were stained with H2A-mcherry and microtubules were stained with Tubulin-GFP. The cells were grown on a cover slip in a culture dish for two days. DMEM media supplemented with 10% fetal bovine serum. Penicillin and streptomycin were used as the growth media. The cells were incubated in a humidified 5% CO2 incubator at ${37}^{\circ }$ C temperature. After 24 h, the cover slip having cells was mounted on a custom-designed mounting stage in a vertical orientation as mentioned above.

All cells were maintained at 37°C and 5% CO₂ under humidified conditions. HeLa cells were obtained from ATCC. Control primary fibroblasts were from Cell Applications, Inc. PINK1 Q456X fibroblasts have been described before [31 (link), 45 ]. HeLa cells and fibroblasts were cultured in Dulbecco’s modified eagle medium (DMEM, Invitrogen) containing 10% FBS (BioWest). Fibroblast medium was supplemented with 1% non-essential amino acid and 1% Penicillin/Streptomycin (both Invitrogen). HeLa cells stably expressing EGFP-Parkin, untagged Parkin, 3xFLAG-Parkin C431S or the EGFP-Parkin and mitoKeima have been described before [16 (link), 21 (link), 25 (link)]. iNeurons were generated as described [16 (link)].

HeLa cells were ordered from ATCC and maintained in RPMI 1640 medium, and HAP1 cells were purchased from Horizon Discovery and maintained in Iscove’s Modified Dulbecco’s medium. The ID8 mouse ovarian cancer cells were kindly provided by Dr. Guang Peng’s laboratory (MD Anderson Cancer Center). HeLa GRB2 deficient cells and HAP1 GRB2 deficient cells were generated by using CRISPR/Cas9 system as described previously^{11 (link)}. ID8 GRB2 stably knocked-down cells were generated by using GRB2 shRNAs which were purchased from Dharmacon (RMM4431-200333332, RMM4431-200335970, RMM4431-200399380, RMM4431-200404712). Media were supplemented with 10% fetal bovine serum (Lonza) and 1% antibiotic/antimycotic (Lonza) except The ID8 cells were maintained in DMEM supplemented with 4% fetal bovine serum (Lonza), 5 μg/mL insulin, 1% penicillin/streptomycin, 5 μg/mL transferrin, and 5 ng/mL sodium selenite. Cells were incubated at 37 °C in a humidified incubator with 5% CO2. Mycoplasma testing of these cell lines has confirmed negative results.