Cxp software v2

After removed the cells with 0.25% trypsin (GIBCO), the cell suspension was rinsed by pre-cooled PBS and then gently re-suspended with 70% ethanol at 4 °C for 12 h. The fixed cells were rinsed with PBS and incubated with propidium iodide containing RNase A at 37 °C for 30 min (Beyotime, C1052). The single cells were screened before flow analysis (Cytomic FC 500 MCL, Beckman Coulter, USA). Data was obtained using CXP software v2.2 (Beckman Coulter, USA), and set the parameter to FL3-620nm BP. 10,000 events were collected per sample manually determined the percentage of G1, G2 and S phases. Data in different groups were imported in Excel software (Microsoft) and generated the graph of relative cell population.
The S phase cells were detected by EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, C0071S). The cells on coverslip were treated with pre-warmed EdU (20 µM) for 2 h, and then fixed by 4 % paraformaldehyde in PBS for 15 min and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Each process followed by PBS washing for 3 times and the cells were incubated in click additive solution (Click Reaction Buffer, CuSO₄ and Azide 488) at room temperature for 30 min. The followed steps of staining the antibodies and nucleus were same as the immunofluorescence assay.

Flow cytometry samples were collected using a Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using CXP software v2.2 (Beckman Coulter, Indianapolis, IN, USA). The following antibodies were purchased from BioLegend (San Diego, CA, USA): PE anti mouse/human CD207, PerCP/Cy5.5 anti-human CD1a, PE mouse IgG1, and PerCP/Cy5.5 mouse IgG1. For intracellular staining, cells were prepared according to the Affymetrix eBioscience protocol. Briefly, single cell suspensions were treated with IC Fixation Buffer, 1X Permeabilization Buffer, and the appropriate antibody, prior to being analyzed via flow cytometry.