Annexin 5 apc pi apoptosis kit

Huh7 cells, which were seeded in 6-well plates, were washed with PBS and stained with Annexin V-APC/PI apoptosis kit (70-AP107-100, Multisciences, Zhejiang, China). Approximately 10⁴ cells were analyzed by a CytoFlex LX (Beckman, CA, USA). Cells were gated based on the forward and scatter characteristics.

To observe the effects of circ-ATXN2 overexpression on cell death, CoCl₂ was used to create apoptotic models of the cells. Vector-GFP- and circ-ATXN2–GFP-expressing cells were seeded at 1 × 10⁵ cells per well in 6-well plates and incubated with 500 μM CoCl₂ for 24 h. Annexin V-APC/PI apoptosis kit (MULTI SCIENCES) was used to determine cell apoptosis. Cells were collected in 500 μl of binding buffer with 3 μl Annexin V-APC and 3 μl PI for 20 min at room temperature in the dark. Apoptotic cells were detected using a flow cytometer (DxFLEX, Beckman Coulter, United States) (He et al., 2021 (link); Song et al., 2021 (link)).

Apoptosis was quantified using an Annexin V-APC/PI Apoptosis Kit (MULTISCIENCES, Hangzhou, China). Briefly, the cells were stained with Annexin V and propidium iodide (PI) after washed with PBS. Flow cytometry was performed using a CytoFLEX™ flow cytometer (Beckman Coulter, Inc., California, USA). Annexin V–PI − , Annexin V–PI + , Annexin V + PI − , and Annexin V + PI + staining represented viable cells, necrotic cells, early apoptotic cells, and late apoptotic cells, respectively.

Gemcitabine (GEM) hydrochloride was obtained from Hansen Pharmaceutical Co., Ltd. (Jiangsu, People’s Republic of China). GEM-HSA-NP was prepared with nanoparticle albumin-bound technology, by which GEM was mixed with HSA in an aqueous solvent and passed under high pressure through a jet to construct drug albumin nanoparticles, as previously reported (29 (link)). Normal saline (NS) was obtained from Shanghai Baxter Healthcare Co., Ltd. (Shanghai, People’s Republic of China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from Bo’ao Biological Technology Co., Ltd. (Shanghai, People’s Republic of China). Cell Counting Kit-8 (CCK8) was purchased from Dojindo (Kyushu, Japan). The Annexin V-APC/PI apoptosis kit was purchased from Multi-Sciences (Hangzhou, People’s Republic of China). The ATP assay kit was purchased from Beyotime Institute of Biotechnology (Shanghai, People’s Republic of China).

Tumor cell digestion was performed as previously described for the isolation of primary RCC cells. After filtering, tumor cells were resuspended in PBS containing 5 × 10^‐3m EDTA and 1% FBS. For measurement of PD‐L1 expression, non‐immune cells were removed via density gradient centrifugation, and the remaining cells were stained with anti‐PD‐L1 (66248‐1‐Ig, Proteintech) or isotype‐control antibodies. After three washes with PBS, cells were stained with Alexa Fluor 488‐conjugated goat anti‐mouse IgG (A‐11001, Invitrogen) for 20 min. For detection of cytotoxic cytokines production, cells were treated with 50 ng mL^‐1 phorbol 12‐myristate 13‐acetate (PMA), 1 × 10^‐6m ionomycin and protein transport inhibitor (BD) for 6 h at 37 °C. Cells were then fixed and permeabilized with a Fixation and Permeabilization Solution Kit (BD, 554714) following the manufacturer's instructions, and cells were then stained with the indicated primary antibodies. For measurement of HLA‐A2 expression, tumor cells were stained with FITC anti‐human HLA‐A2 (343303, Biolegend) or isotype‐control antibodies. For apoptosis analysis, cells were collected and evaluated by the Annexin V‐APC/PI apoptosis kit (AP‐107, Multi Science) according to the manufacturer's instructions.
Samples were analyzed with a Beckman CytoFLEX Flow cytometer (Beckman Coulter, USA), and FlowJo10 software was used to analyze the data.