Trypsin edta solution

HaCaT cells (1 × 10⁶) seeded in 10-cm dish (SPL Life Sciences Co., Ltd., Gyeonggi-do, Korea) and incubated for 24 h were treated with CORM-2 and/or RES for 6 h, irradiated with UVB, and then allowed to recover for 12 h. Afterward, cells were trypsinized (Trypsin-EDTA solution, Welgene, Gyeongsan, Korea), and collected for protein extraction. Cytoplasmic proteins were fractionated by applying NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (NER and CER, Thermo Scientific/Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. Briefly, harvested cells were lysed using CER I and II and then centrifuged at 10,000× g for 30 min at 4 °C. The resulting supernatant was considered as the cytoplasmic fraction and stored at −20 °C. Protein content was determined in both fractions using Bradford assay (Bio-Rad, Hercules, CA, USA) as indicated by the manufacturer. Lastly, lysates were combined with sample loading buffer (40 μg/15 μL buffer) containing 62.5 mM tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue, 5% β-mercaptoethanol, and 20% glycerol, and then rapidly heated at 95 °C for 5 min. The extracts were labeled and stored at 0–20 °C prior to Western blot analysis.

RPMI 1640, PBS buffer (pH7.4) and Trypsin-EDTA solution were purchased from Welgene (Korea). Dynabead-CD45, fetal bovine serum (FBS) and Anti-Pan Cytokeratin (Alexa Fluor 488) were obtained from Invitrogen (MA, USA). Anti-EpCAM was purchased from Abcam (UK). Antibiotic/Antimycotic solution and Ficoll-paque plus were purchased from GE Healthcare (IL, USA). Formaldehyde solution, Bovine Serum Albumin, Triton X-100, and Hoechst 33342 were acquired from Thermo Scientific (MA, USA). Percoll [pH8.5 - 9.5 (25 °C) cell culture tested] was purchased from Sigma-Aldrich (MO, USA), and Anti-CD45 (Alexa Fluor 594) was obtained from BioLegend (CA, USA). ScreenCell Cyto R kit was purchased from ScreenCell (France) and used according to the instructions given by the manufacturer.

Primary astrocytes cultured in a culture dish were reacted with 1x trypsin-EDTA solution (Welgene) at 37°C for 3 min and suspended by pipetting. The suspended astrocytes were then placed in a 15 mL conical tube (SPL) and centrifuged at 2,000 rpm at 4°C for 10 min. The supernatant was removed, and 1 mL of astrocyte culture medium was added, followed by pipetting of single cells. Astrocytes were dispensed with a culture solution on 0.1 mg/mL poly D-lysine-coated cover glass, Ti, and GO-Ti. The astrocytes were then incubated at 37°C and 5% CO₂ for 24 h.

Cell cycle phase distribution was measured by propidium iodide (PI) staining and flow cytometry. Cells were detached from the plate using Trypsin-EDTA Solution (Welgene), washed with DPBS, fixed in 70% ethanol at −20°C for 16 h, washed twice in DPBS, treated with RNase A (100 μg/mL) (BIOFACT, Cat. No. PE290-25h), and stained with 2 μg/mL PI (Thermo Scientific, Cat. No. P3566). Staining profiles indicative of cell cycle stage distribution were measured using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and CytExpert software (Beckman Coulter). Results are presented as content histograms, with number of cells plotted on the y-axis and DNA content as measured by PI fluorescence on the x-axis.

Neural stem cells (NSCs) were obtained from the cortex and hippocampus of embryos on the embryonic day 14.5 fetuses as previously described.
²⁵ The cerebellum, midbrain, olfactory bulb, and blood vessels of the embryo were removed. The cortex and hippocampus tissues were immersed in cold Ca²⁺/Mg²⁺‐free Hanks' balanced salt solution (LB 203‐06; Welgene, Geongsan, Republic of Korea). Next, the tissues were incubated at 37°C for 20 min with DNase‐I (DN25; Sigma‐Aldrich, MO, USA) and Trypsin–EDTA solution (LS‐015‐08; Welgene, Geongsan, Republic of Korea). After stopping the reaction with fetal bovine serum (10082139; Gibco, MA, USA), cells were obtained by centrifugation. Cells were suspended as a single cell in an NSC medium: DMEM/F12 (LM‐002‐05; Welgene, Geongsan, Republic of Korea) supplemented with Penicillin/streptomycin (30010; Hyclone Laboratories, UT, USA), 1% N‐2 supplement (17502048; Gibco, MA, USA), 20 ng/mL EGF (PHG0311; Gibco, MA, USA), 20 ng/mL bFGF (PHG0368; Gibco, MA, USA), and 10% Insulin‐transferrin‐selenium‐sodium pyruvate (51,300,044; Gibco, MA, USA).
²⁵ ,
²⁶ (link) NSCs were seeded into 2 × 10⁷ cells on a 100 mm cell culture dish. After day 3, NSCs were sub‐cultured with a fresh medium for proliferation or differentiation.