Click it plus edu alexa fluor flow cytometry assay kit

Methamphetamine (M8750), LiCl, BIO, and propidium iodide were purchased from Sigma (St. Louis, MO). Access to meth was approved by state and federal regulations. Senescence‐associated β‐galactosidase (SA‐βGal) kit and anti‐GFAP G5 antibody were purchased from Cell Signaling Inc (Danvers, MA). Other antibodies include Iba1 antibody (Fisher Scientific #019‐19741, Pittsburgh, PA), active β‐catenin antibody (US Biological C2069‐47, Salem, MA), total β‐catenin antibody (C2206; Sigma), p16^INK4A antibody (Proteintech #22515‐1‐AP, Chicago, IL), GAPDH antibody (G99445; Sigma), antineurofilament heavy chain (Millipore Ab5539, Danvers, MA). Click‐iT Plus EdU Alexa Fluor Flow Cytometry Assay kit (C10646) was from Invitrogen (Waltham, MA).

Cell cycle and newly synthesized DNA were analyzed based on the methods described before (47 (link), 48 (link)) with some modifications. Briefly, after treatment, cells were washed twice with DPBS and fixed with 75% cold ethanol at 4°C overnight. Afterward, cells were centrifuged at 200 g for 5 min and resuspended with 1 ml DPBS, followed by incubation with 50 µg/ml RNaseA (Invitrogen, USA) (Cat. no. 1692412), and 50 µg/ml propidium iodide (PI) (Life Technologies™, USA) (Cat. no. 3566) for 30 min. For newly synthesized DNA analysis, cells were incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU) and processed according to the manufacturer’s instructions of Click-iT™ Plus EdU Alexa Fluor™ Flow Cytometry Assay Kit (Invitrogen, USA) (Cat. no. 10634) and proceed to the next step for staining the cells for DNA content by PI. To examine the newly synthesized DNA, double staining was conducted using PI with EdU, which was incorporated into DNA during active DNA synthesis and coupled to fluorescent dye that can be detected by different excitation and emission filter from PI (49 (link)). Cell cycle and newly synthesized DNA was determined by flow cytometry (Beckman Coulter FC 500 MPL).