Dmem low glucose

Human multiple myeloma RPMI 8226 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% l-glutamine, and 1% penicillin and streptomycin (Euroclone, Italy). Human glioblastoma U87-MG cells were cultured in Dulbecco's modified Eagle's medium (DMEM) low glucose supplemented with 10% fetal bovine serum, 1% l-glutamine, and 1% penicillin and streptomycin (Euroclone, Italy). Cells were incubated in a humidified incubator at 37°C and 5% CO₂. A stock solution of all compounds in dimethyl sulfoxide (DMSO) (50 mM) has been prepared and then directly diluted in culture medium.

Bone marrow MSCs were purchased from Lonza (Basel, Switzerland) and cultured in a mesenchymal stem cells basal medium bullet kit (Lonza). MSCs were used for the different experiments until passage 6 and expressed the typical MSC markers (CD105, CD29, CD73, CD44 and CD90) (not shown).
MSC-EVs, used as control (EV-CTRL), were obtained by ultracentrifugation, as described in Reference [6 (link)]. Briefly, EVs were obtained from supernatants of MSCs cultured overnight in (Roswell Park Memorial Institute (RPMI) medium. After removal of cell debris and apoptotic bodies by centrifugation at 3000× g for 20 min, EVs were purified by 2 h ultracentrifugation at 100,000× g at 4 °C. EVs from control MSCs or from modified MSCs were used freshly or stored at −80 °C after resuspension in RPMI supplemented with 1% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA).
Murine renal tubular epithelial cells (mTECs) were obtained as previously described [20 (link)] and cultured in Dulbecco’s Modified Eagle Medium (DMEM) low glucose (Euroclone, Pero, Italy) supplemented with 10% fetal calf serum (FCS, Euroclone), penicillin (50 IU/mL), and streptomycin (50 µg/mL) (Sigma). Murine TECs were characterized for positive staining to cytokeratin, alkaline phosphatase and aminopeptidase A, and for negative staining for endothelial (von Willebrand factor), hematopoietic (CD45) and glomerular (nephrin) markers.

HeLa cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) low glucose (Euroclone), supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml Penicillin and 100 μg/ml Streptomycin. SH-SY5Y cells were routinely cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (Gibco), supplemented with 10% FBS, 100 U/ml Penicillin and 100 μg/ml Streptomycin. The day before the experiment, cells were seeded at 10⁵ cells per well in six-well plates or in glass bottom dishes (WillCo-dish) or at 10⁴ cells per well in 4- and 8-well formats Chamber Slides (Lab-Tek). DNA transfection in HeLa cells was carried out using Effectene transfection reagent (QIAGEN) according to manufacturer’s instructions. DNA transfection in SH-SY5Y cells was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) according to manufacturer’s instructions.
SH-SY5Y were differentiated with 10 μM retinoic acid (RA) (Sigma-Aldrich) for 3 days.
Primary hippocampal neurons were obtained from postnatal day (P) 0 B6/129 mice as previously described (Siano et al., 2019 (link)). At 48 h after plating cells have been transfected with Lipofectamine 2000 according to manufacturer’s instructions.

Human multiple myeloma RPMI 8226 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin and streptomycin (Euroclone, Italy). Human glioblastoma U87-MG cells were cultured in DMEM low glucose supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin and streptomycin (Euroclone, Italy). Cells were incubated in a humidified incubator at 37 °C and 5% CO₂. A stock solution of all compounds in DMSO (50 mM) was prepared and then directly diluted in culture medium.

The cell line was derived from the fresh tumor obtained from the surgery performed after 3 courses of neoadjuvant chemotherapy. This was established by mechanical disaggregation of the aseptic surgical sample, after three washes in saline solution. Small fragments of tissue were seeded in 24-well plates at high dilution. The culture medium used was DMEM low glucose (Euroclone) supplemented with 20% fetal bovine serum (FBS, Gibco), 2 mmol/L l-glutamine (Euroclone) and 100 g/mL penicillin-streptomycin (Euroclone). Cells were maintained at 37 °C in an atmosphere containing 5% CO₂. When a confluence of spread cells from fragments of tissue was reached, cells were carefully detached and passed in new flasks. Cells were monitored daily to evaluate growth and morphology, and they were carefully detached and re-seeded upon reaching confluence. The RH30 cell line was maintained in RPMI 10% FBS, 2 mmol/L l-glutamine (Euroclone), and 100 g/mL penicillin-streptomycin (Euroclone), whereas the RD18 cell line was cultured in DMEM high glucose 2 mmol/L l-glutamine (Euroclone) and 100 g/mL penicillin-streptomycin (Euroclone). Cell line authentication was achieved by using short tandem repeat (STR) DNA fingerprinting (Eurofins Medigenomix, Ebersberg, Germany), and cells were regularly tested for mycoplasma-free infection.