Annexin 5 pi

Lipofectamine 2000 and Annexin V/PI were from Invitrogen (Carlsbad, CA); ECL reagents, goat anti-rabbit and anti-mouse secondary antibodies from Thermo (Rockford, IL); protein A/G Plus-Agarose (sc-2003), polyclonal antibodies against RARγ (C-20, sc-551) and Akt1/2/3 (sc-8312), and monoclonal antibodies against Bax (6A7, sc-23959), p85α (B-9, sc-1637), p53 (sc-126), Bcl-2 (sc-509), p-GSK3β (sc-81495), and Cyclin D1 (sc-20044), and FITC-labeled anti-rabbit IgG from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal antibodies against GAPDH (G8795) and β-actin (A5441), Citral (CS:5392-40-25), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4,6-Diamidino-2-phenylindole (DAPI) from Sigma; anti-mouse IgG conjugated with Cy3 from Chemicon international; monoclonal antibodies against pAKT (ser473) (cst-4060) and cleaved caspase-3 (asp175) (#9664), and polyclonal antibody against PARP (#9542) from Cell Signaling Technology (CST); monoclonal antibody against GSK-3β (3D10, ab93926) from Abcam; monoclonal antibody against Ki67 (RB-9043) from Fuzhou Maixin Biotech Co. Ltd., China; PVDF membranes from Millipore; proteinase inhibitor cocktail (11-873-580-001) and PhosSTOP (04-906-837-001) from Roche.

Cells were plated in 6-well plates (2 × 10⁵ cells/well). Cells were treated by cisplatin with 0 or 0.33 μM. The propidium iodide stained the cells after 24 h. The BD Cycle Test Plus DNA Reagent Kit (BD Biosciences, Shanghai, China) has been used in the cell-cycle analysis, following the protocol offered by the manufacturer. The cells were analyzed by FAC scan (BD Biosciences, Shanghai, China), and the percentage of cells in G0/G1, S, or G2/M phase was estimated. Every experiment was conducted at least three times.
Cells were seeded in 6-well plates and resuspended in binding buffer, washed with PBS twice and trypsinized after 48 h. Then, Annexin V/PI (Invitrogen, United States) was used to stain the cells for 15 min in the dark at the room temperature. Then, cell population analysis was conducted by flow cytometry.

After transfection, the cells were seeded overnight at a density of 5×10³ cells in 96-well plates in DMEM containing 10% FBS, and then exposed to various concentrations of gefitinib for 72hours. 10μLof CCK-8 reagent (Dojingdo Molecular Technology, Japan) was added to the cells for 1hour at 37°C, and the absorbance in each well was measured at 450 nm by an enzyme-labeled instrument.
The PC9/R cells were seeded in 6-well plates for 24hours and then transfected with si-UCA1-1 and the negative control. After gefitinib treatment for 72hours, the cells were trypsinized, washed twice with PBS, and resuspended in binding buffer. They were then stained with Annexin V/PI (Invitrogen, USA) for 15min in the dark at room temperature, and the cell populations were analyzed by a flow cytometer.

Mitochondrial membrane potential was measured by fluorescence levels upon staining with JC-1 (Invitrogen, Cat M7514) and MitoTracker Deep Red (Invitrogen, Cat M22426) at 0.5 μg/ml for 30 min at 37°C according to the manufacturer’s instructions. Apoptosis of cells was assessed by staining cells with Annexin V/PI (Invitrogen, Cat V13242) at 37°C for 30 min according to the manufacturer’s instructions. The cells were then washed with PBS and resuspended in cold PBS containing 1% FBS for flow cytometric analyses with Guava easyCyte System 8 (Millipore 25801, Hayward, CA, United States).

Cells were plated in 6-well plates (2 × 10⁵ cells/well). 24 h post transfection of siRNA H19-2 as described above, A549/DDP cells were treated by DDP at a final concentration of 5 mg/L. The propidium iodide stained the cells after 24 h. The BD Cycle Test Plus DNA Reagent Kit (BD Biosciences, Shanghai, China) has been used in the cell-cycle analysis, following the protocol offered by the manufacturer. The cells were analyzed by FAC scan (BD Biosciences, Shanghai, China), and the percentage of cells in G0/G1, S, or G2/M phase was estimated. Every experiment was conducted at least three times.
The A549/DDP cells were seeded in 6-well plates. Afterward, they were transfected through negative control and si-H19-2. These cells were resuspended in binding buffer, washed with PBS twice and trypsinized after 48 h. Then, Annexin V/PI (Invitrogen, USA) was used to stain the cells for 15 min in the dark at the room temperature. Then, cell population analysis was conducted by flow cytometry.

After transfection, the cells were seeded overnight at a density of 5 × 10³ cells in 96-well plates in DMEM containing 10% FBS, and then exposed to various concentrations of gefitinib for 72 hours. 10 μLof CCK-8 reagent (Dojingdo Molecular Technology, Japan) was added to the cells for 1hour at 37°C, and the absorbance in each well were measured at 450 nm by an enzyme-labeled instrument. The PC9/R and PC9/G2 cells were seeded in 6-well plates for 24 hours and then transfected with si-BC087858 and the negative control. After gefitinib treatment for 72 hours, the cells were trypsinized, washed twice with PBS, and resuspended in binding buffer. They were then stained with Annexin V/PI (Invitrogen, USA) for 15min in the dark at room temperature, and the cell populations were analyzed by a flow cytometer.

BMDMs and total lung cells from mice were labeled with the fluorochrome-conjugated primary antibodies to F4/80, MHCII, and CD206 for 30 min. For cell death analysis, cells were digested with 0.05% Trypsin-EDTA (Gibco,Thermo Fisher Scientific), washed with PBS, and then co-stained with Annexin V/PI (V13242, Invitrogen) followed by flow cytometry. Cells were gated on F4/80- and MHCII-positive expression, which were identified as M1 macrophages. Cells were gated on F4/80- and CD206-positive expressions, which were identified as M2 macrophages. Unstained and fluorescein-conjugated isotypic cells were used as controls. Samples were acquired on a flow cytometry analyzer (LSR II; BD Biosciences), and data were analyzed with the DIVA software (BD Biosciences).