Fitc conjugated anti brdu antibody

Logarithmically proliferating MuSCs were washed three times with PBS and changed into fresh medium containing 10 μM BrdU (Sigma-Aldrich, cat# B5002). Two hours later, MuSCs were harvested and fixed with ice-cold ethanol at 4 °C overnight. On the second day, cells were permeabilized with 2 M HCl and 0.5% TRITON X-100 for 30 min at room temperature and washed by 0.1 M Na₂B₄O₇ for 5 min. Next, cells were stained with FITC-conjugated anti-BrdU antibody (Thermo Fisher Scientific, cat# 11-5071-42) and PI for 30 min at room temperature, followed by FACS analysis.
In vivo EdU incorporation assay was performed as previously described.^{86 (link)} Muscles were injected with CTX, and EdU (100 μg/mouse, dissolved in PBS. RIBOBIO, cat# C00052) was injected intraperitoneally twice (48 h and 24 h before sacrifice). After digestion, MuSCs were isolated as the 7-AAD⁻CD45⁻CD11b⁻CD31⁻Sca1⁻Vcam⁺Integrin-α7⁺ population by FACS. MuSCs were then fixed, and EdU was stained with Cell-Light Apollo 567 Stain Kit according to the manufacturer’s instructions (RIBOBIO, cat# C10371-1). The percentage of EdU⁺ MuSCs was analyzed in BD LSRFORTESSA.

To assay apoptosis, A549 cells were transfected with miR-630 mimic or CDC7 siRNA and harvested for analysis 48 h after transfection by trypsinization, and then stained with Annexin V/PI Apoptosis Detection Kit (Biosea, Beijing, China) according to manufacturer's instructions. Cell apoptosis were analyzed with a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The fluorescence signals of apoptosis cells were represented by Annexin⁺/PI⁻ and Annexin⁺/PI⁺. For BrdU incorporation, cells were labeled with 50 μM BrdU for 1 h. Cells were washed in PBS, fixed in ice-cold 70% ethanol for 24 h and later DNA was denatured with 2 N HCl for 30 min. Incorporated BrdU was detected using FITC-conjugated anti-BrdU antibody (11-5071; eBioscience, San Diego, CA, USA).