Cell light edu

DNA synthesis was assessed by the Cell-Light EdU (5-ethynyl-2'-deoxyuridine) Apollo488 In Vitro Imaging Kit according to the manufacturer’s instructions (RiboBio).

DNA synthesis was assessed using the Cell-Light EdU (5-ethynyl-2′-deoxyuridine) DNA Cell Proliferation Kit (RiboBio Co) according to its instruction. Images of the cells were captured with a fluorescence microscope (Nikon, Tokyo, Japan). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to count the fluorescent points.

Astrocyte proliferation was determined using the Cell-Light EdU (5-ethynyl-2′-deoxyuridine) Apollo567 In Vitro Kit (#C10310; Riobio, Guangzhou, China) following the manufacturer’s instructions. SB216763 or Nec-1 were added to the medium during reoxygenation and the astrocytes were incubated for 24 h. Cells were sequentially incubated with 20 μM EdU during reoxygenation; 4% paraformaldehyde (to fix the cells for morphological analysis) for 30 min at room temperature; and 0.5% Triton X-100 (to permeabilize the cells) for 10 min. Finally, 1× Apollo reaction solution was added and the cells were incubated for 30 min. To label GFAP, cells were exposed overnight to an anti-GFAP antibody (1:1,000, #53-9892-82, Thermo, MA, USA) at 4°C. Hoechst (1:5,000; #33258; Sigma-Aldrich, MO, USA) was used to stain the nuclei. Images were obtained using a confocal microscope (LSM 710; Carl Zeiss, Jena, Germany).