T75 cell culture flask

Conditioned medium was generated like the supernatants for the secretome analysis. Briefly, 1.2 × 10⁶ senescent or non-senescent cells were seeded per T75 cell culture flask (Greiner AG, Kremsmünster, Austria). The following day, 20 mL fresh medium was added to each flask and 24 h later, the conditioned medium was transferred to a centrifuge tube (Greiner AG, Kremsmünster, Austria), centrifuged at 522×g for 5 min and the supernatant filtered with 0.2 µm syringe filters (VWR International, Radnor, USA) on SOFT-JECT^® syringes (Henke-Sass Wolf, Tuttlingen, Germany). The filtered conditioned medium was divided in aliquots and stored at − 80 °C.
To assess the influence of conditioned medium on the migration of healthy MSCs, the IncuCyte migration tool was used as described above. Therefore, only untreated low-passage MSCs were seeded in the insert plate and regular cell culture medium or conditioned medium was added to the reservoir plate. To reduce the potential problem of nutrient deficiency, also a mixture of 50% conditioned medium with 50% cell culture medium was used. The plates were recorded every 2 h for 188 h in total. The experiment was performed with 8 wells per condition and three times independently.

Rat vascular endothelial cells (RVECs; ATCC, Manassas, VA, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Corning) supplemented with 10% v/v fetal bovine serum (FBS; Fisher Scientific, Pittsburgh, PA, USA), 0.2% v/v of 250 μg/mL Amphotericin B (GE Healthcare), and 0.1% v/v of 50 mg/mL Gentamycin sulfate on T-75 cell culture flasks (Greiner Bio-One, Monroe, NC, USA) under static conditions at 37 °C with 5% CO₂ (HERAcell 150i; Thermo Scientific, Waltham, MA, USA) until they reached 90% confluency. They were re-suspended at a final concentration of 2 × 10⁶ cells/mL.
MCF7 were maintained and cultured in the same manner as culturing RVECs, where DMEM was replaced with Eagle’s Minimum Essential Medium (Corning) and 0.01 mg/mL human recombinant insulin (Sigma-Aldrich) was additionally added. MCF7 was patterned in the same manner as patterning RVECs.

Gli36dEGFR glioma cells were obtained from Dr. David Louis (Molecular Neurooncology Laboratory, Massachusetts General Hospital, Boston, MA) 26 (link),27 (link). The cells were engineered to express the luciferase reporter system (Gli36dEGFR-LITG) as previously described 28 (link). Glioma cells were carefully cultured and observed. Tumor cells displayed the typical growth patterns and phenotypes in vitro and in vivo. The cells were not further genetically authenticated. Thawed cells were cultured in T-75 cell culture flasks (Greiner Bio One, Germany) as adherent monolayer in DMEM (life Technologies) supplemented with GlutaMax, 10% heat inactivated FCS (Invitrogen) and 1% penicillin/streptomycin (PAA Laboratories) at 37°C in a humidified incubator maintained at 5% CO₂ prior to intracranial implantation.

hTERT immortalized human adipose-derived mesenchymal stem cells (hASCs/TERT1, Evercyte) were either used as purchased or retrovirally infected with green-fluorescent proteins (GFP-hASCs) according to the protocol by Knezevic et al. (2017)^{54 (link)}. Endothelial Cell Growth Medium-2 (EGM-2) was prepared by supplementing the EBM-2 Basal Medium with the EGM-2 SingleQuots Supplements from the EGM-2 Endothelial Cell Growth Medium-2 BulletKit (Lonza). hASCS/ GFP-hASCs were cultured in T-75 cell culture flasks (Greiner Bio-One) in EGM-2 supplemented with 10% fetal calf serum (FCS, Sigma-Aldrich). Human umbilical vein endothelial cells (HUVECs, PELOBiotech GmbH) were infected with red-fluorescent protein (RFP)-expressing lentiviral particles at passage 1. Zeocing resistant RFP-HUVECs were selected and cultured in EGM-2 supplemented with 5% FCS in T-25 cell culture flasks (Greiner Bio-One). All cell lines were cultivated in an incubator (BINDER) at 37 °C with 5% CO2 in a humid atmosphere and split at around 80 percent confluency. The media were changed every 2 to 3 days.

The culture of 3T3-L1 cells was performed in T75 cell culture flasks (658 175, Greiner, Frickenhausen, Germany) in growth medium (90% DMEM, high glucose (61965-026, Fisher Scientific GmbH, Darmstadt, Germany), 10% fetal calf serum (FCS) (S1810-500, Biowest, Nuaille, France), 1% penicillin–streptomycin (15140-122, Life Technologies GmbH, Darmstadt, Germany), and 1 mM sodium pyruvate (11360-070, Fisher Scientific GmbH)). Cells were split in a 1:10 ratio every 2 to 3 days. The utilized cultured cells were in passages 14–18.
For NAD(P)H-FLIM measurements, 240,000 cells per well were seeded into 6-well plates (657160, Greiner) in regular growth medium. The growth medium was exchanged daily. After 3 days, the medium was aspirated and replenished with imaging medium (90% DMEM, high glucose, HEPES, no phenol red (21063-029, Fisher Scientific GmbH), 10% FCS (S1810-500, Biowest)). During imaging cells were kept on 37 °C.