Fc500 flow cytometry

Ang II and candesartan were used to stimulate hepatoma cells inoculated in six-well plates for 48 h, while NS administration was set as a control. The cells were collected with trypsin after the termination of the drug action by phosphate buffer washing, and then the rabbit anti-human AT1R primary antibody (Abcam; Cambridge, UK) and FITC-labeled fluorescent secondary antibody (#ZF-0311; ZSGB-BIO; Beijing, China) were added in turn. The percentage of AT1R positive HCC cells was detected and analyzed by FC500 flow cytometry (Beckman Coulter; Brea, CA, USA) and FlowJo software.

For cell cycle analysis, transiently transfected U87 and U251 cells were cultured in six-well plates. All cells were harvested by trypsinization after 48 h, washed with ice-cold PBS two times, and then fixed with 75% ethanol at 4 °C overnight. The cells were resuspended in 200 μL PBS with 5 μL RNase and 10 μL propidium iodide (PI) (Beyotime Institute of Biotechnology, Shanghai, China) for 25 min in the dark at room temperature. Cell cycle distribution was evaluated by FC500 flow cytometry (Beckman Coulter, Brea, CA, USA).
A cell apoptosis assay was carried out by using Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s recommendations. Briefly, U87 and U251 cells transfected with siRNA1-RACK1, siRNA2-RACK1, or si-NC were harvested after 72 h for apoptosis analysis. A total of 195 μL of binding buffer, 5 μL of Annexin V-FITC, and 3 μL of propidium iodide (PI) were added to the suspension, and cells were incubated for 10 min at room temperature in the dark. The cells were immediately examined by flow cytometry (Beckman Coulter, Atlanta, GA, USA). All experiments were conducted independently at least three times.

Cell Cycle and Apoptosis Analysis Kit (Beyotime) was used to measure the cell cycle. Twenty thousand cells were analyzed per sample by FC500 flow cytometry instrument equipped with CXP software (Beckman Coulter). The proportions of G0/G1, S and G2/M cells were calculated and compared with the ModFit LT software.

CaSki and C33A cells were seeded into 6-well plates at a density of 1 × 10⁶ cells per well and exposed to 10 mM metformin with or without 20 μM everolimus for 48 h. Cells were then collected and fixed with 70% ice-cold ethanol at −20 °C overnight. After fixation, cells were centrifuged at 400× g for 10 min at 4 °C, washed with cold PBS, and stained with 0.5 mL PI/RNases staining buffer (PI, 10 μg/mL; RNases, 300 μg/mL; #550825, BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature in the dark. Cell apoptosis was detected using an FITC Annexin V apoptosis detection kit (#556547, BD Biosciences); cells were double-stained with 5 μL FITC Annexin V (20 μg/mL) and 5 μL propidium iodide (PI, 50 μg/mL) for 15 min at RT in the dark. Finally, the stained cells were analyzed using FC500 flow cytometry and CXP software (version 2.3; Beckman Coulter, Brea, CA, USA), and early and late apoptotic or necrotic cells were assessed.

Cells were seeded into a 6-well plate (0.5–1 × 10⁶/well) and incubated overnight. Then cells were treated with 7.5 μg/mL ZC-14 for 6, 12, 18 and 24 h and stained by FITC Annexin V apoptosis Detection Kit I (#556547, BD Pharmingen™, Franklin Lakes, NJ, USA) according to manufacturer’s protocol. Briefly, cells with different treatments were harvested by centrifugation (600× g, 5 min) and washed twice with ice-cold phosphate buffered saline (PBS). After resuspended with 100 μL 1× binding buffer, cells were stained by 5 μL Annexin V-FITC and 5 μL PI in the darkness at room temperature for 15 min. After that, another 400 μL of 1× binding buffer was added, and the samples were determined by FC500 flow cytometry (Beckman, S. Kraemer Boulevard Brea, CA, USA). The cells with 7.5 μg/mL harmine treatment for 24 h were used as the negative control and 0.5 × 10⁵ cells of each samples were used for analysis.

Reprogrammed iPS cells were collected separately by digestion with TripLE solution (Invitrogen). We used 2% paraformaldehyde containing 0.05% Triton X-100 to fix and obtain permeabilized cells. After that, we stained the iPS cells with rabbit or mouse anti-human Oct4, Nanog, Sox2, SSEA-4 and TRA-1-60 antibodies (Stemgent, San Diego, CA) for analysis on FC500 flow cytometry (Beckman, Fullerton, CA) using CXP analysis software (Beckman). We assumed rabbit or mouse isotypic antibodies as negative controls respectively.

The phenotype of macrophages was identified by analyzing the surface markers of macrophages using flow cytometer. Briefly, macrophages were harvested, washed, and resuspended in 100 µl PBS solution at a density of 5 × 10⁶ cells/ml. For Thp1 macrophages, cells were incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and PE-conjugated CD163 antibody (12-1639-42, eBioscience, San Diego, CA, USA) for 30 mins at 37°C. For Raw264.7 macrophages, cells were incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz) and PE-conjugated CD206 antibody (141705, Biolegend, San Diego, CA, USA) for 30 mins at 37°C. For phenotype analysis of primary macrophages extracted from mouse 4T1-Luc xenografts, cells were incubated with CD45-PE-Cy7 (25-0451-82, eBioscience), FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz) and PE-conjugated CD206 antibody (141705, Biolegend) for 30 mins at 37°C. After incubation, cells were washed once with PBS and subjected to analysis using a FC500 flow cytometry (Beckman Coulter, Fullerton, CA, USA) or a FACSAria III flow cytometer (BD Biosciences, San Diego, CA, USA). The F4/80⁺/CD163⁺ subpopulation, the F4/80⁺/CD206⁺ subpopulation or the CD45⁺/F4/80⁺/CD206⁺ subpopulation were quantified and defined as M2 phenotype macrophages.