Annexin 5 pi staining

The CS, CePO₄/CS, and CePO₄/CS/GO scaffolds were added to MDA-MB-231 cell culture medium, and the cells were exposed to radiation from a near-infrared (IR) spectrometer with a power density of 4.6 W cm⁻² for 3 min. The apoptotic cells were detected using flow cytometry (annexin V/PI staining) according to the protocol provided by the manufacturer (BD Bioscience, USA). The RAW264.7 cells were then added to the CS, CePO₄/CS, and CePO₄/CS/GO scaffolds. The cells with the CePO₄/CS/GO scaffolds were divided into two groups; one group was exposed to NIR radiation, and other was not. According to the protocol provided by the manufacturer (BD Bioscience, USA), the polarization of the macrophages was examined by flow cytometry using anti-mouse CD16/32-PE (cat. No. 553145) and anti-mouse CD206-Alexa 647 (cat. No. 565250).

The percentages of cells in the different phases of cell cycle were evaluated by determining the DNA content after propidium iodide (PI) staining (BD Biosciences). Briefly, LOVO and RKO cells (4 × 10³) were harvested 48 h and incubated in PBS containing RNase (1 mg/mL) for 10 min at room temperature. Finally, samples were stained with propidium iodide (1 mg/mL) for 30 min at 4°C. Data acquisition was done by flow cytometry (FACSCalibur, BD Biosciences) using Cell Quest software. Annexin V/PI staining (BD Biosciences) and flow cytometry analysis were performed according to the manufacturer's instruction. Briefly, LOVO, RKO and SW620 cells were incubated with Annexin V FITC and PI prior to analysis using a flow cytometer.

Cell cycle progression and apoptosis induction using Annexin V-PI staining (BD Biosciences) were measured as previously described [28 (link)]. Caspase 3/7 apoptosis activity was measured using Incucyte software (Essen Biosciences) as previously described (Braggio, Cancer, 2019). Briefly, apoptotic index was measured by dividing the fluorescence of caspase 3/7 substrate by total number of cells measured using Vybrant® DyeCycle™ Green stain (Life Technologies). Data were analyzed using Incucyte software (Essen Biosciences).

For Annexin V/propidium iodide (PI) immunofluorescence, HUVECs were pre-treated directly with DMEM added with drugs and MSU crystals for 24 h. Meanwhile, RAW264.7 macrophages were pre-treated with DMEM containing Kin (6.25–25 μg/ml) and Indo (20 μg/ml) and stimulated with MSU (300 μg/ml) for 24 h. The CM supernatants of macrophages were collected to further administer with HUVECs for 24 h. Cell apoptosis levels of HUVECs from both administrations were determined with flow cytometry via Annexin V/PI staining according to the manufacturer's instructions (BD Bioscience).^{67 (link)} The percentages of apoptotic HUVECs were presented.
To measure the MSU crystal uptake by macrophages, RAW264.7 macrophages were pre-treated with DMEM containing Kin (6.25–25 μg/ml) and Indo (20 μg/ml) and stimulated with MSU (300 μg/ml) for 24 h. Cells were then fixed in 4% paraformaldehyde and side-scatter high analysis was performed to determine the crystal uptake in macrophages according to previous report.^{18 , 68}

Distribution of cell cycle phases were determined by PI nuclear staining and flow cytometry. Cells were seeded and treated after 24 hr with Control (0.1% DMSO), 5 μM or 10 μM GSK126. After 72 hr cells were harvested, washed and stained with PI staining solution. Samples were evaluated using a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed using Modfit software. Cell death was determined using Annexin V/PI staining according to the manufactures instructions (BD) and analyzed by flow cytometry with CellQuestPro software.