Annexin 5 fitc pi staining kit

The number of apoptotic and necrotic cells was counted using an Annexin V-FITC/PI staining kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were seeded in a 12-well plate at a density of 1×10⁵/well and then incubated in 5% CO₂ atmosphere at 37°C for 48 h. Aβ_1–42 at 10 μM was added in each well. Different concentrations of 3F5, IgG and PBS were added to the wells. IgG was considered as a negative control. SH-SY5Y cells were washed at 48 h later with cold PBS and collected by centrifugation at 92 g for 5 min. Subsequently, the cells were stained with a combination of fluorescein annexin V-FITC and PI for 10 min at room temperature. Apoptosis was examined with by flow cytometry (ACEA Biosciences, Santiago, USA).

Cells were incubated in 6-well plates and exposed to the indicated concentrations of PAC or vehicle control for 24 h. Apoptosis and cell cycle analyses were performed using an Annexin-V FITC/PI Staining Kit and Cell-Cycle Detection Kit (KeyGEN-Biotech, CN) following the manufacturer's instructions. All data were evaluated by FlowJo software (BD Biosciences, USA).

An annexin V-FITC/PI staining kit (Nanjing KeyGen Biotech Co., Ltd.) or an annexin V-APC/7-aminoactinomycin D (7-AAD) staining kit (MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, Zhejiang, China) was used to evaluate apoptosis. Following chaetocin treatment, cells were collected, washed and stained in working solution (500 µl binding buffer with 5 µl annexin V-FITC and 5 µl PI or 500 µl binding buffer with 5 µl annexin V- APC and 10 µl 7-AAD) for 15 min at room temperature in the dark. The FACSCanto II flow cytometry (BD Bioscience) was used to detect apoptotic cells. Annexin V-FITC⁺ and annexin V-FITC⁺-PI⁺ cells or annexin V-APC⁺ and annexin V-APC⁺7-AAD⁺ cells were considered apoptotic cells.

Analysis of cell cycle was carried out by FACS according to the manufacturer's instructions using Cell Cycle and Analysis Kit (Keygen, KGA512). Cells were fixed with 70 % ethanol overnight at 4 °C, then centrifuged at 1000g for 5 min to remove the ethanol followed by PBS washes. The samples were then treated with RNase A for 30 min at 37 °C, and stained with propidium iodide (PI) then analyzed by the flow cytometer. Apoptosis was measured using the Annexin V-FITC/PI staining kit (Keygen, KGA101) according to the manufacturer's instruction. Briefly, after 48 h or 72 h treatment of drugs, breast cancer cells and lung cancer cells were re-suspended in 500 μl of binding buffer. Annexin V-FITC (5 μl) and PI (5 μl) were added into 100 μl cell suspension containing 1×10⁵ cells. Samples were mixed gently and incubated at room temperature for 15 min, shielded from light. Finally, the percentage of apoptotic cells were measured by flow cytometry (BD Biosciences), and the Flowjo software was used for FACS data analysis.

Apoptosis was then detected with annexin V-FITC/PI staining kit (Nanjing KeyGen Biotech). NKYS, SNK6 cells were treated with normal conditions, MTX (20 mM), gemcitabine (5 μM), or combination treatment for 24-48 hr, respectively. The cells were then collected, washed, and stained in a working solution (500 µl binding buffer with 5 µl annexin V-FITC and 10 µl PI) for 15 min at room temperature in the dark. The FACS CytExpert 2.0 (Beckman Coulter) was used to detect and analyze apoptotic cells. Annexin V-FITC ⁺ and annexin V-FITC ⁺-PI ⁺ cells were considered as apoptotic cells.

Cellular apoptosis was quantified by flow cytometry using an Annexin V-FITC/PI Staining kit as per the manufacturer's instruction (KeyGEN BioTECH, Jiangsu, China). Briefly, 5 × 10⁵ cells were harvested and suspended in 500 μl of binding buffer containing 5 μl annexin V-FITC and 5 μl PI. These cells were incubated for 15 min at room temperature in the dark. The apoptotic cells were detected by the FACSCalibur (BD Biosciences).

Drug-induced apoptosis was detected by FACS analysis using an Annexin V-FITC/PI staining kit (KeyGEN BioTECH, China). RKO and LoVo seeded into six-well plates were cultured overnight. After treatment with Cisplatin alone or Aspirin alone or their combination for 48 hours, cells were collected and washed once with cold PBS, subsequently stained with FITC-labeled Annexin V and PI. The stained cells were analyzed by flow cytometry (BD Accuri™ C6).