Dead cell kit

Percentage of viable cells was evaluated by trypan blue dye exclusion method. After monolayer cell trypsinization (0.25 mg/ml trypsin, Gibco, Thermo Fisher Scientific), cells were stained with trypan blue (0.04%, Gibco) for 2 min, and vital, unstained cells were counted with emacytometer. The percentage of unstained cells was obtained as mean ± SD of four independent experiments. 786-O cell apoptosis was assayed with MuseAnnexinV and Dead Cell kit (Millipore, Darmstadt, DE); 1 × 10⁵ cells from untreated, DMSO and CPTH2 samples were centrifuged (2000 rpm, 5 min), washed in PBS, and resuspended in PBS plus 1% BSA (Sigma-Aldrich) 1% FBS, with 100 μL of Dead Cell reagent containing AnnexinV and 7-aminoactinomycin D (7-AAD). After 20 min RT incubation at room temperature in the dark, cells were applied to a Muse Cell Analyzer (Millipore), and the results are expressed as percentage of apoptotic cells ± SD.

Detroit 562 human squamous carcinoma cell line originating from pharynx primary location was used in the present study and purchased from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). Detroit 562 HNSCC cells were seeded on 6-well microplates and were cultured in standard culture medium (EMEM; Eagle's Minimum Essential Medium) containing 10% fetal bovine serum (FBS; Pasching, Austria) and 1% penicillin-streptomycin (PAA Laboratories GmbH, Pasching, Austria) at 37°C in 5% CO₂ in air (CO₂ incubator, Heraeus Instruments, Hanau, Germany). Additionally, cells were cultured with 100 μg/mL streptomycin, 100 IU/mL penicillin, and 0.25 μL/mL amphotericin B at 37°C in a 5% CO₂ atmosphere. Reagents were purchased from PAA Laboratories GmbH (Pasching, Austria); caffeic acid and caffeic acid phenethyl ester were purchased from Sigma (St. Louis, MO, USA). Muse™ Annexin V and Dead Cell kit were purchased from Millipore (Billerica, MA, USA).

The distribution of apoptotic cells was analyzed using a Muse^TM Annexin V and Dead Cell Kit (Millipore Co., Billerica, MA, USA), based on the manufacturer’s protocol. NHDF cells were harvested following irradiation with 55 mJ/cm² of UV and subsequent treatment with MEH or MED for 24 h. The harvested NHDF cells were suspended in DMEM, and 100 μL of this cell suspension (1 × 10⁴ cells/mL) was incubated with Muse^TM Annexin V and a Dead Cell Kit (Millipore Co., Billerica, MA, USA) reaction reagent for 20 min at room temperature. Finally, cells were analyzed using a Muse^TM Cell Analyzer (Millipore Co., Billerica, MA, USA). After gating based on size, cells were distinguished into four different groups: non-apoptotic cells [Annexin V (-) and 7-AAD (−)], early apoptotic cells [Annexin V (+) and 7-AAD (−)], late apoptotic cells [Annexin V (+) and 7-AAD (+)], and mostly nuclear debris [Annexin V (+) and 7-AAD (+)]. These data are represented as the number of live and apoptotic cells.

The extent of apoptosis was determined by Muse Annexin V and a dead cell kit (Millipore, Billerica, MA, USA). According to the manufacturer’s procedure, A549 and H460 cells (10 × 10⁴ cells/well) were seeded in 6-well plates and treated with CTT (0, 5, or 10 μM) or GF 20 μM for 24 h. After being harvested from the culture medium, cells were washed with 1 mL PBS and 100 μL of Muse™ Annexin V Dead Cell reagent were added to them. Next, cells were incubated at room temperature for 20 min without light. The apoptosis was then analyzed with Muse cell analyzer and Muse analysis software (EMD Millipore).

We performed the annexin V assay for the measurement of apoptosis using Muse Annexin V and Dead Cell Kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. We analyzed the relative percentage of total apoptotic cells using a Muse Cell Analyzer (Millipore, Billerica, MA, USA).

Human hepatocellular cancer cell line (HepG2) was purchased from American Type Culture Col-lection (ATCC; Rockville, MD, USA). Blueberry leaves were bought from Nanjing Jinrui Blueberry Professional Cooperative. Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin-ethylene diamine tetraacetic acid (EDTA) solution, fetal bovine serum (FBS), and penicillin-streptomycin solution were acquired from GIBCO (Grand Island, NY, USA). 3-[(4,5)-Dimethylthiazol-2-yl]-2,5-diphenyl tetra-zolium bromide (MTT), bovine serum albumin (BSA), phosphate buffer saline (PBS), sodium palmitate and Oil Red O and were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Deionized water was obtained from a Milli-Q Gradient Water System (Millipore Corp., Bedford, MA, USA). In addition, The Muse TM Annexin V and Dead Cell Kit was bought from Merck Limited (Darmstadt, Germany). Bradford reagent was obtained from Bio-Rad Laboratory (Hercules, CA, USA). RIPA lysis buffer, antibodies against caspase-3 and Bcl-2 were bought from Cell Signaling Technology Inc. (Boston, MA, USA), β-actin and secondary antibody were provided by Abcam Inc. (Cambridge, UK).

Apoptotic cells were analyzed using Muse Annexin V and Dead Cell Kit (Merck Millipore) according to the previously described protocol (11). This assay utilizes Annexin V to detect phosphatidylserine on the external membrane leaflet of apoptotic cells and dead cell marker, 7-AAD, as an indicator of cell membrane integrity. Briefly, 1 × 10⁵ cells were resuspended in culture medium containing 1% FBS and incubated with Muse Annexin V and Dead Cell Reagent for 20 min at room temperature in the dark. Cells were quantified using Muse Cell Analyzer and Muse analysis software.