Apoptosis detection kit

An apoptosis detection kit (Promega, Madison, WI) was used to detect apoptosis according to a previously described method [14 (link)]. In brief, renal sections were subjected to TUNEL staining in accordance with the manufacturer’s instructions. Later, IF microscopy was used to analyze the samples using a Zeiss Axiovert 200 M fluorescent microscope equipped with an AxioCamMR3 camera. Six fields (magnification 400×) were randomly selected from every section from 10 different rats, and cells with positive TUNEL staining were analyzed.

In this study, the apoptosis induced by plant extracts was determined by measuring the activity of caspases 3 and 7 using an apoptosis detection kit (Promega, G8091). The Caspase-Glo^® 3/7 assay was performed according to the manufacturer’s protocol. First, PC-3 and LNCaP cells were seeded in 96-well plates at 2.5 × 10³. The cells were left for 24 h to allow cell attachment. After 24 h, the cells were treated with extracts, fractions, vehicle control (DMSO), paclitaxel, and a blank (cell-free medium) for 48 h. Next, 100 μL of Caspase-Glo^® 3/7 reagent was added to each well after 48 h of incubation and mixed gently for 30 s. Then, the plate was incubated for 1 h at room temperature. After the completion of the incubation period, the luminescence of each plant extract was measured using an Infinite^® M200 plate-reading luminometer (Tecan, Männedorf, Switzerland). The assay was performed independently in triplicate, and the results were calculated using the following equation:

The TUNEL assay was performed with cells cultured in chamber slides using the apoptosis detection kit (Promega) according to the manufacturer’s instructions. Slides were evaluated under a microscope (Olympus IX71) with a digital camera (Retiga 1300) interfaced to a computer with PCI software. The apoptotic cells were quantified as follows. The number of cells of the area (DAPI staining) were captured and counted. There were approximately 100 cells per area and three areas were captured in each slide. The fluorescence-labeled cells (apoptotic cells) from the same area were captured and counted. Three independent experiments were performed and analyzed.

To count intra-hepatic nodules, the tumor-bearing livers were dissected, fixed with 10% neutral buffered formalin overnight, embedded in paraffin, and sectioned into 4 μm slices. Sections were stained with hematoxylin and eosin (H&E) and subjected to immunohistochemical analysis. For frozen blocks, tumor specimens were dissected from mice, embedded immediately in ornithine carbamoyl transferase (OCT; Miles, Elkhart, IN, USA) compound, snap frozen in liquid nitrogen and stored at −70°C. Frozen tissues for CD31/PECAM-1 and/or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining were prepared and fixed in cold acetone. The TUNEL assay was performed with a commercially available apoptosis detection kit (Promega, Madison, WI, USA) with some modification. Immunohistochemistry procedures were performed and all antibodies and agents for immunohistochemistry were purchased from sources as described previously [24] (link). Control samples exposed to secondary antibody alone showed no specific staining. The stained sections were examined under an Olympus BX51 microscope (Olympus) equipped with an Olympus DP71 12.5 megapixel digital microscope camera.

Apoptosis of RTECs was measured using annexin V and propidium iodide kit. Apoptosis was reflected by annexin V positive ratio (%), which was determined using flow cytometry (BD FACSVerse, San Jose, CA) with methods described earlier (Wang et al., 2013 (link)) and a same gating strategy.
TUNEL staining of rat kidney tissue was carried out using a Promega apoptosis detection kit. Immunofluorescence for TUNEL staining was performed using FITC and 4′,6-diamidino-2-phenylindole (DAPI) and the glass was mounted with cover slips and imaged under a confocal microscope (LSM 780; Carl Zeiss, Oberkochen, Germany). TUNEL-positive apoptotic cells were counted in 10 random high-power fields (HPF; 300 cells each). Data are expressed as the number of apoptotic cells/HPF (magnification: 400×).

For detection of apoptotic cells, TUNEL staining was carried out using a Promega apoptosis detection kit. Immunofluorescence for TUNEL staining was performed with fluorescein isothiocyanate (FITC). The glass was mounted with cover slips containing 4′,6-diamidino-2-phenylindole (DAPI) and imaged under a Confocal Microscope (LSM 780; Carl Zeiss, Oberkochen, Germany). Cells stained green fluorescence and DAPI stained blue fluorescence were detected as TUNEL-positive apoptotic cells. Both TUNEL-positive and DAPI-positive cells were counted in 10 random high-power fields (HPF; 300-cells each). Data are expressed as the number of apoptotic cells/HPF (400x magnification).