Annexin 5 fitc pi apoptosis detection kit

The cells were washed twice and centrifuged after detached by EDTA-free trypsin solution (Gibco, USA). 100 µl of Binding Buffer (1×) was added to resuspend the cell pellets. 5 µl of Annexin V-FITC and 10 µl of PI (AnnexinV-FITC/PI Apoptosis Detection Kit, Meilunbio, China) were added and con-incubated at 37 °C for 15 min at dark. The FITC channel (excitation wavelength 490 nm, emission wavelength 525 nm) and the PerCP channel (excitation wavelength 535 nm, emission wavelength 615 nm) were selected. A double-negative control group (unstained sample) was used to adjust the voltages of the Forward Scatter (FSC) and the Side Scatter (SSC), and two single-positive control groups (samples stained with FITC and PI separately) were used as compensation controls to compensate for the spectral overlap. A quadrant gate was set, and four regions were defined (Q1: Annexin V-/PI-, Q2: Annexin V+/PI-, Q3: Annexin V+/PI+, Q4: Annexin V-/PI+). The overall apoptosis rate (early and late stages) of each group was recorded.

Bupleurum chinense DC roots, produced in Dingxi (place in Gansu), harvested in late September, were dried, crushed, and passed through an 80 mesh sieve to obtain Bupleurum chinense DC powder, which was placed in a desiccator and sealed for storage (relative humidity was 70 ± 5%, the temperature was 25 ± 5 °C). Standards of monosaccharides and 5-fluorouracil were purchased from Sigma (St. Louis, MO, USA). T-series dextran was acquired from Solarbio (Beijing, China). Sephadex G-150 was purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). A JC-1 mitochondrial membrane potential detection kit was gained from Solarbio Science & Technology Co., Ltd. (Beijing, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). The cell cycle analysis and apoptosis kit were obtained from Beyotime Biotech (Nantong, China). All other chemical reagents and materials were of analytical grade.

The treated cells were digested with trypsin, washed with PBS, and collected by centrifugation. Cell staining was performed using an Annexin V-FITC/PI Apoptosis detection Kit (Dalian Meilun, China) according to the manufacturer’s instructions, and a fluorescence microscope (Olympus, Japan) was used to photograph them.

Cell apoptosis was quantified by the Annexin-V-FITC/PI Apoptosis Detection Kit (Meilunbio, Dalian, China). After the intervention, AR42J cells were digested in Accutase (Mutisciences, Hangzhou, China) and collected subsequently. Having been washed by D-PBS and resuspend in the Annexin V Binding Buffer, 100 μL of cell suspension was transferred into the test tube for staining. 5 μL of Annexin-V-FITC and 5 μL of PI solution were added to each sample, then incubated for 15 min at room temperature in the dark. Having been added 400 μL Annexin V binding buffer to the test tube, cell suspension was texted by the flow cytometry (BD FACSAria™ II Sorter, USA). The results were quantified by FlowJo-V10.0 software.

Distearoyl-sn-Glycero-3-Phosphoethanolamine- PEG2000-Hyaluronan (DSPE-PEG2000-HA) was purchased from Ruixi Biological Technology (Xian, China). UA, lecithin, cholesterol, coumarin 6 (C6), a Mitochondrial Membrane Potential Assay Kit with JC-1, Reactive Oxygen Species Assay Kit, and Annexin V-FITC/PI Apoptosis detection kit were purchased from MeilunBio (Dalian, China). Antibodies against p53, ARTS, Bcl-2, Bax, Caspase 3, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abcam (Baston, USA). A549 cells were purchased by m the Cell Bank of the Chinese Academy of Sciences (China).

4-Cyano-4-(dodecylsulfanylthiocarbonyl) sulfanyl pentanoic acid (DCT), N,N′-dicyclohexylcarbodiimide (DCC), acetone oxime, monomethoxy PEG (Mw = 5000 Da), and 2,2′-azobis(2-methylpropionitrile) (AIBN) were purchased from Sigma–Aldrich. 2-Hydroxyethyl disulfide was purchased from Macklin, and 4-(N,N-dimethylamino) pyridine (DMAP) was purchased from Heowns. Triphosgene, methacryl chloride, N-ethylethanolamine, propyl bromide, and camptothecin were obtained from Energy Chemical. 5(6)-TAMRA cadaverine (Rho) was purchased from Haoran Biological Technology Co., Ltd. Cyanine 5.5 amine was purchased from Lumiprobe. 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime Biotechnology Co., Ltd. Cell counting kit-8 (CCK-8) was purchased from Beijing Solarbio Technology Co., Ltd. An Annexin V-FITC/PI apoptosis detection kit was purchased from Meilun Biological Technology Co., Ltd. (Dalian, China). 1-(4-(Aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c] quinoline-4-amine (IMDQ) was provided by Nanjing Aikon Chemical Ltd. Anti-CD11c, anti-CD86, anti-CD80, anti-CD40, anti-GrB, anti-TNF-α, anti-CD3e, anti-IFN-γ, anti-MHC-I, anti-MHC-II, anti-CD103, anti-F4/80, anti-CD206, anti-iNOS, anti-CD4, anti-CD8, anti-Foxp3, anti-CD44, anti-CD62L were purchased from Biolegeng (American).