Accuri c6 plus flow cytometry

To measure IFN-γ levels by stimulating splenocytes in vitro, splenocytes were cultured in 12-well plates containing proteins of rPoAMA-1. After 18 h, the cells were simultaneously mixed with various concentrations of phorbol 12-myristate 13-acetate (50 ng/mL PMA; Sigma), ionomycin (1 µg/mL; Solarbio), and brefeldin A (5 µg/mL; Solarbio) for 6 h at 37 °C with 5% CO₂. The cells were washed twice with PBS. Subsequently, 50 µL PBS containing CD4–488 (1:200; BioLegend, California, USA) and CD8-APC (1:300; BioLegend, California, USA) were added to samples at 4 °C for 1 h, and then centrifuged (1500 × g, 10 min) and washed twice with PBS. Then, 100 µL of fixative (BioLegend, California, USA) was added, and the samples were incubated in a dark environment at 4 °C for 20 min. The samples were then washed with membrane washing buffer (BioLegend, California, USA), and 50 μL of IFN-r-PE (1:200; BioLegend, California, USA) diluted in transmembrane washing solution was added in a dark environment at 4 °C overnight. Finally, the samples were analyzed in BD Accuri C6 PLUS flow cytometry (BD, New Jersey, USA).

The cell apoptosis was determined by Annexin V-PE/7-AAD (Becton Dickinson Co, NJ, USA) according to the instructions of the manufacturer. Approximately, 1 × 10⁵ H9c2 cells were placed on six-well plates. Followed by the two washes with PBS, the H9c2 cells were collected and analyzed using BD Accuri® C6 Plus Flow Cytometry (BD Bioscience, NJ, USA).

The Annexin V-APC/propidium iodide (PI) apoptosis detection kit (KeyGEN, China) was used to detect apoptosis by flow cytometry. DLBCL cells were cultured in vitro and divided into nine groups, including untreated (blank control), negative control, Astragali Radix group, Glycyrrhizae Radix et Rhizoma group, Ginseng Radix et Rhizoma group, Pseudostellariae Radix group and Codonopsis Radix group. After cultivation for 48 h, cells were washed with PBS, resuspended in 100 µL binding buffer at a concentration of 2 × 10⁶ cells/ml, and anti-Annexin V APC-conjugated antibody and PI were added. The mixtures were incubated for 15 min at room temperature, supplemented with binding buffer to 500 µL and processed by BD Accuri C6 Plus Flow Cytometry (BD Biosciences, United States). Data were analyzed through flow cytometry (BD PharmingenTM, United States).

MCF-7 cells were cultured for 24 h at a density of 2 × 10⁶ cells per 10 cm dish (SPL Life Sciences). Cultured cells were exposed to THMGT at concentration of 3 mg/ml for 3, 6, and 9 h. Annexin V/PI staining was performed according to FITC Annexin V Apoptosis Detection Kit II (BD) protocol. Stained cells were analyzed by BD Accuri C6 Plus Flow cytometry (BD Biosciences). Results were determined as follows: viable cells expressed (−) Annexin V/(−) PI, necrotic cells were (−) Annexin V/(+) PI, early apoptotic cells were (+) Annexin V/(−) PI, and late apoptotic cells were (+) Annexin V/(+) PI.

MCF-7 cells were cultured for 24 h at a density of 2 × 10⁶ cells per 10 cm dish (SPL Life Sciences). Cells were subsequently treated with 3 mg/ml THMGT for 3, 6, and 9 h. Cells were then collected in Trypsin/EDTA, fixed in cold 70% (v/v) ethanol, and stored at −20°C for at least 24 h. After washing two times with PBS, cells were incubated with 0.2 mg/ml RNase for 30 min at 37°C and subsequently stained with 10 μg/ml propidium iodide staining buffer for 15 min at room temperature. DNA content was analyzed by BD Accuri C6 Plus Flow cytometry (BD Biosciences).