Edu assay edu staining proliferation kit ifluor 488

The EdU assay to measure genomic DNA synthesis was conducted using the EdU assay/EdU Staining Proliferation Kit (iFluor 488) (abcam; 219801) following the manufacturer’s protocol. Briefly, monocytes, macrophages, and positive control 293T cells were incubated with EdU for 2 h. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min, permeabilized, and labeled with the iFluor 488 dye. Unstained cells and cells that were fixed prior to EdU incubation were used as a control for fluorescent background. Flow cytometry was conducted using a FACSymphony A5 (BD Biosciences) and analyzed using FlowJo software (BD Bioscience; https://www.bdbiosciences.com/en-us/products/software/flowjo-v10-software). Cell debris and doublets were excluded from iFluor 488 analysis. iFluor 488 gating was established on unstained cell populations.

EdU proliferation assays were performed using the EdU Assay/EdU Staining Proliferation Kit (iFluor 488) (Abcam, ab219801) according to the manufacturer's protocol. Isolated OPCs were proliferated for 4.5 days and incubated with EdU for 2 h. The cells were fixed with 4 % PFA and followed by immunofluorescence staining for Ki67. An additional EdU labeling step with the EdU reaction mix, including iFluor 488 azide, was performed between the secondary antibody incubation and the DAPI counterstaining step, with an incubation period of 30 min at RT.