Penicillin streptomycin

For AT-MSCs isolation, the previously reported protocols [23 (link), 24 (link)] were adjusted as follows: abdominal subcutaneous adipose tissue biopsy samples were thoroughly washed with Hanks’ solution (BioloT, Russia) supplemented with Diflucan (Pfizer, USA), gentamicin (50 μg/ml, PanEco, Russia), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia). Each sample was minced into small (~ 1 mm³) pieces with scissors and transferred into conical tubes. A fourfold volume of 0.1% collagenase type I solution (PanEco, Russia) in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 supplemented with 2 mM l-glutamine (BioloT, Russia) and 50 mg/ml gentamicin was added to each tube, and enzymatic dissociation was performed for 20 min at 37 °C. Enzymatic digestion was blocked by adding threefold volume of DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% fetal bovine serum (FBS) (Thermo Fisher, USA), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia). The tubes were then centrifuged at 100g for 10 min at room temperature and the supernatant was discarded. The cell pellet was resuspended in DMEM/F12 supplemented with 2 mM l-glutamine (BioloT, Russia), 10% FBS (Thermo Fisher, USA), and penicillin/streptomycin (100 U/ml/100 µg/ml, PanEco, Russia) and seeded on Petri dishes.

IR-780 iodide (Sigma-Aldrich, Saint Louis, MO, USA), Nile Blue A perchlorate (Sigma-Aldrich, Saint Louis, MO, USA), Poly(D,L-lactide-co-glycolide) (RG 858 S, Poly(D,L-lactide-co-glycolide) ester terminated, lactide:glycolide 85:15, Mw 190,000–240,000 Da, Sigma, Darmstadt, Germany), PVA (Mowiol 4-88, Sigma, Steinheim, Germany), chitosan oligosaccharide lactate (5 kDa, Sigma, Steinheim, Germany), penicillin/streptomycin (Paneco, Moscow, Russia), 2 mM L-glutamine (Paneco, Moscow, Russia), DMEM/F12 with 2 mM L-alanyl-L-glutamine (Gibco, Paisley, UK), Hoechst33342 (Thermo Fisher Scientific, Waltham, MA, USA), Versene solution (Paneco, Moscow, Russia), penicillin-streptomycin (Paneco, Moscow, Russia), bovine serum albumin (Paneco, Moscow, Russia), Herceptin (Roche, Mannheim, Germany), Crystal violet dye (LenReaktiv, St. Petersburg, Russia), fetal bovine serum (HyClone, Logan, UT, USA), Carboxy-H₂DCFDA, (Thermo Fisher Scientific, Waltham, MA, USA), and resazurin sodium salt (AlfaAesar, Lancashire, UK).

Neuronal cells (20,000) were plated in each well of Matrigel-treated 24-well plates and were cultured in DMEM/F12 medium supplemented with 2% serum replacement (“Gibco”, Carlsbad, CA, USA), 1 mM non-essential amino acids (“Paneco”, Moscow, Russia), 2 mM L-glutamine (“ICN Biomedicals Inc.”, Hackensack, NJ, USA), penicillin–streptomycin (50 U/mL; 50 µg/mL) (“Paneco”, Moscow, Russia), 1% B27 supplement (“Life Technologies”, Carlsbad, CA, USA), 5 µM forskolin (“Stemgent”, Cambridge, Massachusetts, USA), 20 ng/mL BDNF, 20 ng/mL GDNF and 200 µM ascorbic acid (all from “Peprotech”, Cranbury, NJ, USA) in a CO₂ incubator at 5% CO₂ and 37 °C. On the day of the experiment, the growth medium was changed to DMEM/F12 medium supplemented with 2 mM L-glutamine (“ICN Biomedicals Inc.”, Hackensack, NJ, USA) and penicillin–streptomycin (50 U/mL; 50 µg/mL) (“Paneco”, Moscow, Russia). N-ADA or N-DDA (5 µM) were added to the cells at the same time point and the control cultures were treated with an equal volume of DMSO. Cells were then incubated for 40 min and treated with 200 µM H₂O₂; the same volume of growth medium was used for the control wells. At 3 and 24 h time points of incubation, the cells were collected for RNA extraction and preparation of cell lysates and the conditioned growth media were collected for ELISA.