Annexin 5 fitc

Colonies from the CFA were isolated from soft agar via dilution in warm PBS and centrifugation. Single cells were obtained via incubation with Accutase, Merck Millipore, Billerica, MA, USA, for 15 minutes at 37°C, subsequently washed in PBS and resuspended in 1x- Annexin V binding buffer. Cells were incubated for 15 minutes with Annexin V-FITC, Santa Cruz Biotechnology, Dallas, TX, USA, (0.1–1μg). Shortly before FACS analysis, propidium iodide was added at appropriate dilutions and samples were analyzed on a FACS Calibur, BD.

Quantification of apoptosis was performed by measuring cell surface accumulation of phosphatidylserine (PS) via binding of annexin-V-FITC (Santa Cruz Biotechnology) using flow cytometry. Briefly, treated cells were washed with PBS, centrifuged and resuspended in annexin-V binding buffer (in mM: 10 HEPES, pH 7.4; 140 NaCl; 2.5 CaCl₂) supplemented with 0.2 μg/μl annexin-V-FITC. After 15 min of incubation cell were centrifuged and resuspended in the same buffer containing 0.6 μg/ml of propidium iodide (PI). Samples were analyzed in duplicate (15,000 cells/condition) in a MACSQuant Analyzer 10 apparatus (Miltenyi Biotec) using 488 nm light for excitation. FITC signal was detected at 518 nm and PI at 620 nm. Data are presented as percentage of cells undergoing early apoptosis (annexin-V-positive, PI-negative cells) or late apoptosis (annexin-V and PI-positive cells).

B16F10 cells (2 × 10⁵) were seeded in 6-well culture plates and incubated at 37°C for 24 h. Cells were treated for 24 h with jara (0.4 and 0.8 μM) and jari (0.2 and 0.4 μM); control cells were treated with PBS alone. Cells found in the supernatant and adherent cells were incubated with specific binding buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl₂, pH = 7.4), containing 5 μL of annexin V-FITC (Santa Cruz, USA) and 1.8 ug/mL propidium iodide for 30 min at room temperature in the dark. After incubation, 400 μL binding buffer was added and cells were analyzed with FACScalibur (Becton DicKinson) using CellQuest software, determining the percentage of apoptotic cells.

BV-2 cells (2.5 × 10⁵/well) were seeded in 6-well plates, treated with ribavirin and LPS as described above. Assessment of cell viability involved double staining of cells with Annexin V-FITC (Santa Cruz, Dallas, Texas, USA) and propidium iodide (PI; BD Pharmingen, San Diego, CA, USA). Annexin V binds to phosphatidylserine, exposed on the surface of early apoptotic cells, while PI uptake is marker for necrotic or later apoptotic cell death. Negative staining for both dyes was characteristic of viable cells. Staining was performed according to the manufacturer's instructions. Flow cytometry was conducted on CyFlow Space Partec (Partec GmbH, Munster, Germany) and the data was analyzed using PartecFloMax software (Partec GmbH, Munster, Germany).

For cell cycle analysis, AGS cells were treated with TIIA or DMSO as control for 48 hr. TIIA treatment concentrations were 0.625 μM, 1.25 μM, 2.5 μM, and 5.3 μM; 0.1% DMSO dissolved in culture medium was used as control. After treatment, cells were harvested with trypsin/EDTA, fixed with 70% ethanol, then spun down, after which ethanol was removed. Then each sample was mixed with RNase A (100 μg/mL), incubated at 37°C for 1 hr, and stained with propidium iodide (PI) (Santa Cruz Biotechnology, Inc.) at a concentration of 100 μg/mL in the dark at room temperature for 15 min. For apoptosis analysis, AGS cells were treated with TIIA or DMSO control medium for 48 hr after 24 hr of seeding (3.5 × 10⁵ cells in 10-cm plates). TIIA treatment concentrations were 1.25 μM, and 5.3 μM; 0.1% DMSO dissolved in culture medium was used as control. After treatment, cells were harvested with trypsin/EDTA, suspended, and counted. Then each sample was adjusted to a concentration of 10⁶ cells/tube and stained with Annexin V-FITC (Santa Cruz Biotechnology, Inc.) and PI (Santa Cruz Biotechnology, Inc.) dissolved in binding buffer (Santa Cruz Biotechnology, Inc.) in the dark at room temperature for 15 min. Both cell cycle distribution and apoptotic cells proportion were then analyzed with a BD FACSCanto II flow cytometer (BD Biosciences) and FCS Express 4 (BD Biosciences).

Zein Protein (Merc, Germany), alcalase enzyme, lecithin, Tween 80, cholesterol, ethanol, RPMI, RNase A, and yellow tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St Louise, IL). Fetal bovine serum and DMSO were provided by Thermo (Waltham, MA), PBS, trypsin/EDTA, propidium iodide (PI), Triton X100, and DCFDA/H2DCFDA Cellular ROS Assay Kit were obtained from Abcam (Cambridge, UK). Penicillin, streptomycin, and Tris-HCl buffer were purchased from GeneDirex, Inc. (Gongye Rd, Taiwan). Annexin V-FITC (EXBIO, Czech), Bax, Bcl-2, caspase-3, -8, -9, and p53 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).