Annexin 5 fitc pi detection kit

Cell death process was detected by the Annexin V-FITC/PI Detection Kit (KeyGEN, China) followed by flow cytometry analysis. SYTOX Orange (Invitrogen, USA) staining was used to detect dead cells. Cell death was monitored by LDH release assay using the LDH Cytotoxicity Assay Kit (Beyotime, China) according to manufacturer’s instructions.

Different concentrations of Matrine combined with Osthole (high, medium, and low) were added after infection with 10^4.4 TCID₅₀ of PCV2 for 2 h. Cells were cultured for 48 h and then collected for staining with Annexin V-FITC/PI detection kit (Keygen Biotech, Nanjing, China). The samples were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Apoptosis was measured using an Annexin V-FITC/PI detection kit (KeyGEN BioTECH, Nanjing, China) following kit’s protocol. Briefly, cells were digested with non-EDTA trypsin. After washing with PBS, the cells were resuspended with binding buffer mixed with FITC-conjugated Annexin V and PI for 15 min at room temperature. After washing, the signal was measured by flow cytometry (BD Biosciences; San Jose, CA, USA) with excitation and emission wavelengths at 488 nm and 530 nm.

Polyethylene glycol methyl ether (MPEG, Mn = 2000, Fluka, USA), ε-caprolactone (ε-CL, Alfa Aesar, USA), stannous octoate (Sn(Oct)₂, Sigma, USA), doxorubicin (DOX, Sigma), methyl thiazolyl tetrazolium (MTT, Sigma, USA), Dulbecco’s modified eagle medium (DMEM Gibco, USA), fetal bovine serum (FBS, Gibco, USA), alginate sodium (Sigma, USA), fluorescein isothiocyanate-dextran (FITC-dextran, Sigma, USA), Annexin V-FITC/PI Detection kit (keyGEN Biotech, China), tricaine (Sigma, USA), 1-phyenyl-2-thiourea (PTU, Sigma, USA), and methanol (HPLC grade, Fisher scientific, UK) were used without further purification. HK was purchased from Suzhou NeuPharma Co. All the materials used in this article were analytic reagent (AR) grade and used as received.
C6 glioma cells were maintained and grown in DMEM supplemented with 10% FBS in a 37 °C incubator with a humidified 5% CO₂ atomosphere.
Tg(flk:EGFP) transgenic zebrafish were provided by Dr. Shuo Lin (UCLA, USA). Female BALB/c nude mice (6–8 weeks old) were purchased from the Laboratory Animal Center of Sichuan University (Chengdu, China). Animal experiments were performed according to the guidelines of the Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China) and approved by the Animal Care and Use Committee of Sichuan University.

Apoptosis was detected using an Annexin-V FITC/PI detection kit according to the manufacturer’s directions (KeyGEN, Nanjing, China). The cells were digested with 0.25% trypsin, washed with ice-cold PBS and resuspended in binding buffer (5 × 10⁵  cells/mL). Then, the cells were centrifuged at 1000g for 5 min at 4 °C. After the supernatant had been discarded, 500 μL of binding buffer, 5 μL of annexin-V-FITC and 5 μL of propidium iodide were added to the cell suspension. After mixing gently, the suspensions were incubated for 15 min at room temperature without light. Finally, the cells were analyzed by flow cytometry (BD LSRII; BD Biosciences).

Cell apoptosis was determined using an Annexin V-FITC/PI Detection Kit, in accordance with the manufacturer’s protocol (KeyGEN, China). The indicated cells were seeded in six-well plates at a density of 30% and were treated with different doses of compound 7594-0035. After 48 h, the cells were stained with Annexin V-FITC and PI and then analyzed by flow cytometry.