Alexa fluor 488 annexin 5 kit

Analysis of SMMC7721 cell apoptosis was done using the Alexa Fluor 488 Annexin V Kit (Invitrogen). In brief, SMMC7721 cells (1 × 10⁶ cells/mL) were washed with phosphate buffered saline (PBS) and centrifuged, and the pellet was resuspended in an Annexin binding buffer and incubated with annexin V and propidium iodide working solutions. Apoptosis was determined on a flow cytometer (BD Biosciences, San Jose, CA, USA) and evaluated by FlowJo software (Tree Star, San Carlos, CA)^{17 (link)}.

Apoptosis was analyzed by Annexin V-fluorescein isothiocyanate staining using an Alexa Fluor 488 Annexin V kit (Invitrogen). In total, 1.0 × 10⁶ of IFITM1—pcDNA3.1 construct-transfected cells, as well as the same amount of pcDNA3.1 vector-transfected cells, were washed twice with cold PBS. Cells were then resuspended in 1 × Annexin-binding buffer for 30 min. Cell apoptosis was determined by Annexin V-fluorescein isothiocyanate/PI double staining. Cell transfection was analyzed on FACS calibration with the software Cell Quest (Becton–Dickinson). All experiments were carried out in triplicate.

To study the cell apoptosis and its role in EC tube formation impairment, HMVE cells were cultured either under normal or ischemic condition with or without ALKBH5 silencing. Apoptotic cell death was determined by flow cytometry using the Alexa fluor 488 Annexin V Kit (# V13241, Invitrogen) following the manufacturer's protocol. Briefly, after siALKBH5 silencing and ischemic stress, around 0.5 million cells were harvested, washed with PBS buffer and incubated with Annexin V Alexa fluor488 (Alexa488) and propidium iodide for 30 min in the dark. The cells were then analyzed by fluorescence-activated cell sorting (FACS) using the FACS Attune NXT instrument and FlowJO software at the UAB Comprehensive Cancer Center core facility. One-way ANOVA (GraphPad Software Inc.) with post-hoc Tukey test was used to calculate statistical significance.

For analysis of apoptosis using Alexa Fluor 488 Annexin V Kit (Invitrogen), the cells (1 × 10⁶ cells/ml) were harvested, washed with PBS and centrifuged at 1000 rpm for 5 min. Then, the cell pellet was re-suspended in the annexin-binding buffer, incubated with annexin V and PI working solution for 15 min at room temperature. Cell apoptosis was determined using FACS caliber Flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, San Carlos, CA).

CD133 Alexa Fluor 488-conjugated Antibody (Novus Biologicals) and EpCAM FITC Conjugated Antibody (Absin Bioscience Inc.) were used to detect the proportion of positive HCC cells. Following sublethal heat stress, HCC cells (1 × 10⁶ cells/ml) were seeded into 6-well plate, incubated with HSC-CM for 48 h. The cells were collected, washed with stain buffer (BD Biosciences), re-suspended in 100 μl stain buffer containing CD133 or EpCAM antibody for 30 min at 4 °C, respectively. Followed by washing in stain buffer and incubating with secondary antibodies for 30 min, FCM was used for analysis using an excitation wavelength at 488 nm filter for fluorescent detection.
After exposure to sublethal heat stress, HCC cells were plated into 6-well plate, incubated with 100 ng/ml POSTN for 48 h and followed by the treatment of 3.3 μg/ml cisplatin for another 24 h. For analysis of apoptosis using Alexa Fluor 488 Annexin V Kit (Invitrogen), cells (1 × 10⁶ cells/ml) were harvested, washed with PBS and centrifuged at 1000 rpm for 5 min. Subsequently, cell pellets were re-suspended in annexin-binding buffer, incubated with annexin V and PI working solution for 15 min at room temperature. Cell apoptosis was measured using FACS caliber Flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, San Carlos, CA).

The effect of ATRA on cell apoptosis was also determined using an Annexin V-Alexa Fluor 488 kit (Invitrogen, OR, U.S.). H9c2 cells cultured on 6-well culture plates were pre-treated and exposed to different concentrations of ATRA or a DMSO equivalent to ATRA at the highest concentration for 24 h which served as a negative control, or 1 mM H₂O₂ which served as an apoptosis-positive control, for 2 h. After H/R, the H9c2 cells were collected and then washed two times in ice-cold PBS. After cells were centrifuged at 300 x g for 5 min, the pellets were resuspended in Alexa Fluor 488-annexin V and propidium iodide binding buffer, mixed gently, and then incubated for 10 min on ice in the dark. Then samples were analyzed at 494 nm excitation wavelength using fluorescence-activated cell sorting (FACS) and a FACSAria I Cell Sorter (Becton-Dickinson, CA, U.S.). Alexa Fluor 488 emission was detected using a 520 nm band pass filter, and a > 600 nm filter was used to detect propidium iodide. The relative number of Annexin-V-positive cells in the H9c2 cells populations was measured using FACSDiva software (Becton-Dickinson, CA, U.S.). The experiment was performed in triplicate.

To induce apoptosis of lung tumor cells (H460), the cells (5×10⁶ cell) were grown overnight and then treated with etoposide (50 μM) (Sigma) in serum-free media for 22 hrs, followed by washing with phosphate-buffered saline (PBS) and binding buffer (10 mM Hepes (pH 7.4), 150 mM NaCl, and 2 mM CaCl₂). The apoptosis was validated using an annexin V-Alexa Fluor 488 kit (Invitrogen) and propidium iodide (PI) for FACS analysis. Normal or apoptotic cells (1×10⁵ cells/ml) were incubated with serial concentration (2, 10, 20, 50 μg/ml) of peptides or proteins in binding buffer (10 mM Hepes (pH 7.4) and 150 mM NaCl, with or without 2 mM CaCl₂) at 4°C for 1 hr. The cells were washed with binding buffer for three times and immediately subjected to FACS analysis (FACScan, Becton Dickinson, San Jose, CA, USA).