Versene

HaCaT (immortalized non-tumorigenic human keratinocytes) (Table S1), A431 (human epidermoid carcinoma cells), human keratinocytes (passage 2 and passage 3), and HeLa (human adenocarcinoma cells) were kindly provided by the Cell Culture Collection for Biotechnological and Biomedical Research, Koltsov Institute of Developmental Biology, Russian Academy of Sciences. The cells were grown in DMEM (Dulbecco Modified Eagle’s Medium; PanEco, Moscow, Russia) (HaCaT and A431) or DMEM/F-12 (human keratinocytes and HeLa) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA), 2 mM L-glutamine (PanEco), 80 μg/mL gentamicin (Belmedpreparaty, Minsk, Belarus), and 0.2% Defined Keratinocyte-SFM Growth Supplement (Thermo Fisher Scientific) (only for human keratinocytes) under standard conditions (37 °C, 5% CO₂). The cells were collected from the surface of a plastic flask with a 1:3 mixture of trypsin solution (PanEco) and Versene (0.2% EDTA in phosphate buffer) (PanEco) and plated on glass cover slips or on Petri dishes with glass bottom and grown for 48 h. Then, GA or DTT was added, and cells were incubated for 24 h.

Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA), Bicuculline methochloride, L-NAME hydrochloride, Glycine, Domoic acid, (R)-(+)-Methanandamide, L-arginine, Acetyl-L-carnitine chloride, UK 14304, Telenzepine dihydrochloride (Tocris Bioscience, UK); Hank’s Balanced salt solution (HBSS), neurobasal medium, B-27 Supplement, fetal bovine serum, fetal calf serum (Gibco); penicillin-streptomycin solution (Dalhimfarm, Russia); 0.1% poly-l-lysine; L-glutamine, L-glutamate, NBQX hydrate, (+)-MK-801 hydrogen maleate, NAAG, Betaine monohydrate, L-carnitine hydrochloride, P-F-HHSiD (p-Fluorohexahydro-sila-difenidol hydrochloride), Methoctramine hydrate, Nystatin (Sigma-Aldrich, USA); KCl (Chimmed, Russia); embryo calf serum (MP Biomedicals, USA); 4% gentamicin (Dalhimfarm, Russia); versene (Paneco, Russia); Fura-2 AM (Invitrogen, USA).
All animal studies were performed in accordance with the legal requirements and were approved by the Animal Ethic Committees of both institutes.

MCF-7 cells were cultured in a CO₂ incubator for 48 h; then, the incubated cells were removed from the flask surface using a solution of 0.25% Trypsin (MP Biomedicals, Santa Ana, CA, USA)/Versene (0.2% EDTA in phosphate buffer, PanEco, Russia) (1:4). Next, the cells were pelleted and washed in 0.01M phosphate-buffered saline (PBS; PanEco, Russia) by centrifugation (at 150 G, 5 min, +37.0 ± 1.0 °C) on a Z 216 MK microcentrifuge (Hermle Labortechnik GmbH, Wehingen, Germany). After removal of the supernatant, the cell pellet was resuspended in 300 μL of PBS. A suspension of viable cells at a concentration 5 × 10⁴ cells/mL was therewith obtained.
The samples intended for imaging were prepared by translocating the suspension of viable cells into a zone restricted by a thin layer of silicone grease on the reflecting surface of a mirror slide; then, the zone containing the cells was covered with a coverslip, whereupon the samples were immediately examined by LIM.