Hoechst 33342

Phenylmethanesulfonyl fluoride (PMSF), McCoy’s 5A medium, Dulbecco’s Modified Eagle Medium (DMEM), tris-base, sodium chloride, bovine serum albumin (BSA), TRIzol^® reagent, tween 20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and sodium azide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from Unionward (Taipei, Taiwan). Trypsin-EDTA, penicillin/streptomycin and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Recombinant RNasin ribonuclease inhibitor, oligo d(T)21 and M-MLV reverse transcriptase were purchased from Promega (Madison Avenue, New York City, NY, USA). 4′,6-diamidino-2-phenylindole (DAPI), lysis buffer, stabilization buffer and Hoechst 33,342 were purchased from ChemoMetec A/S (Gydevang, Allerod, Denmark). CYTO-ID^® autophagy detection kit was purchased from Enzo Life Sciences (cat. no. ENZ-51031; Madison Avenue, NY, USA). Chemiluminescence (ECL) kit was purchased from Bio-Rad (cat. no. 1705061, Hercules, CA, USA).

Activity of the SA-βgal was analyzed by cytochemical staining modified from Dimri et al. [22 (link)], using Senescence β-galactosidase staining kit (Cell Signaling Technology, Leiden, The Netherlands) according to the manufacturer´s instruction. Cytochemical staining was analyzed by manual counting of X-gal-positive cells of random image sections per well. Images were taken on a light microscope (TE2000-S, Nikon, Vienna, Austria). Automated analysis of SA-βgal activity was carried out using the NucleoCounter^® NC-3000™ (Chemometec) following the protocol FlexiCyte^TM. Cells were stained with 33 µM C₁₂FDG (Setareh Biotech, LLC, Eugene, OR, USA) in EGM-2 for 2.5 h at 37 °C, 5% CO₂ adapted from Debacq-Chainiaux et al. [35 (link)]. After staining, cells were washed with PBS and trypsinized. The cell pellet was resuspended in PBS supplemented with 10 µg/mL Hoechst-33342 (Solution 15, Chemometec). The procedure was adapted to the limited cell numbers of the different conditions for MT-SVF adherent. Incubation at 37 °C for 15 min followed, prior to loading in an NC-Slide A2. Fluorescence intensities were displayed in the NucleoView™ software as scatter plots and histograms and were further analyzed in the Plot Manager for defining the C₁₂FDG-positive cell populations.

Apoptotic cells were detected using the CF488A Annexin V and PI Apoptosis kit (Biotium, Fremont, CA, USA) and Hoechst 33342 (ChemoMetec). After staining, the cells were analyzed using an image flow cytometry assay (NucleoCounter NC-3000, ChemoMetec). NucleoView NC-3000 was then employed for automated image flow cytometry analysis.

Growth medium RPMI 1640, trypsin/EDTA and fetal bovine serum were purchased from Cytogen (Zgierz, Poland). A growth medium M-254 and human melanocytes growth suplement-2 (HMGS-2) were acquired from Cascade Biologics (Portland, OR, USA). NC-Slides (A2 and A8), Via-1-Cassette (containing acridine orange and DAPI), Solution 3 (1 μg/mL DAPI, 0.1% triton X-100), Solution 5 (400 μg/mL VitaBright-48, 500 μg/mL propidium iodide, 1.2 μg/mL acridine orange), Solution 7 (200 μg/mL JC-1), Solution 8 (1 μg/mL DAPI), Solution 15 (500 μg/mL Hoechst 33342), and Solution 16 (500 μg/mL propidium iodide) were obtained from ChemoMetec (Lillerød, Denmark). Annexin V-CF488A conjugate and Annexin V binding buffer were obtained from Biotium, Inc. (Fremont, CA, USA). Ketoprofen—KTP (chemical name: (RS)-2-(3-benzoylphenyl)-propionic acid), amphotericin B, penicillin, and dimethyl sulphoxide (DMSO) were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Other chemicals were from POCH S.A. (Gliwice, Poland).

Image cytometry with an NC-3000 Image Cytometer (ChemoMetec, Allerod, Denmark) was used for cellular and mitochondrial phenotyping. A minimum number of 5000 events were analyzed in each experiment. The following assays were executed according to the manufacturer’s recommendations: cell count (acridine orange and DAPI (ChemoMetec)), mitochondrial membrane potential (JC-1 and DAPI (ChemoMetec)), thiol redox status (VitaBright-48, Propidium iodide, and acridine orange (ChemoMetec)). Mitochondrial superoxide levels (MitoSOX™ (Thermo Scientific) and Hoechst-33342 (ChemoMetec)) were measured as described earlier [43 (link)]. To measure mitochondrial mass, 100 nM fluorescent dye MitoTracker™ Green FM (Invitrogen, Carlsbad, CA, USA) diluted in Hank’s balanced salt solution (HBSS) (Invitrogen) was added to cells for incubation for 30 min at 37 °C. After removing excess dye, the cells were collected and a 1:1000 dilution of RedDot2 (Biotium, Fremont, CA, USA) (concentration not specified) was added to each sample before measurement. The NucleoView NC-3000 software (ChemoMetec) was used for data analysis.

Cortical organoids, primary fetal neurons, and astrocytes were manually dissociated and resuspended in Annexin V binding buffer (Invitrogen). Next, Annexin V-CF488A conjugate (Biotium, Inc., Hayward, CA, USA) was added, followed by Hoechst 33342 (Chemometec). After a PBS wash, the cells were resuspended in Annexin V binding buffer (Invitrogen) containing 10 μg/mL propidium iodide (Chemometec). Cells were loaded into NC‐Slide A2 chambers, and the “Annexin V Assay” was run with the NucleoCounter NC-3000.