Pe active caspase 3 apoptosis kit

The skins were stored in formaldehyde 15% for 48 h. Furthermore, alcohol was used as a dehydration agent with concentration of 70%, 80%, and 96%. Xylol was used for clearing process and continued making paraffin block with 60°C of the temperature. The skin tissue that has received paraffin blocks then sliced using a microtome machine and then transferred into a water bath before being placed on a glass object. Immunohistochemistry staining was used primary antibody caspase-3 anti-rat (PE Active Caspase-3 Apoptosis Kit BD Pharmingen^™ with CAS number 550914) and CTL (ATCC^® PCS-800-017^™) for 1 h in 27°C. The dilution was given 10 µl for caspase-3 and 0.1 ml for CTL test. Caspase-3 was executor primary antibody [17 ]. Then, the specimen washed in phosphate buffered saline (PBS) with a pH of 7.4 for 3 times every 5 min. The next preparations were added streptavidin-horseradish peroxidase for 60 min in 27°C and washed in PBS with pH 7.4. Then, the specimens were added chromogen 3,3-Diaminobenzidine tetrahydrochloride for 20 min and washed with aquadest for 5 min.

Caspase-3 activity was determined using PE active caspase-3 apoptosis kit (BD Pharmingen, San Jose, CA, USA). Briefly, MCF-7 (5 × 10⁵) cells in 10-cm dishes were subjected to drug treatment as indicated for 72 h and were re-suspended in 0.5 mL Cytofix/Cytoperm solution for 20 min on ice. Cells were then incubated in 100 μL of Perm/Wash buffer containing 20 μL caspase-3 antibodies for 30 min at room temperature. Each sample was then added to 400 μL Perm/Wash buffer, and caspase-3 activity signals were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA)

Active caspase–3 staining protocol was performed following manufacturer’s instructions (PE active caspase–3 apoptosis kit, BD biosciences, San diego, CA, United States). Total 5 × 10⁵ cells of MCF-7 treated with different concentrations of OXY were harvested and transferred into 15 ml centrifuge tubes and then the suspension was centrifuged at 300 × g for 5 min at 4°C. Cells were washed twice with 1 ml ice–cold PBS and then re–suspended in 0.50 ml BD Cytofix/Cytoperm™ solution. The cells were incubated on ice for 20 min. After that the buffer was removed, the cells were washed twice with 0.50 ml BD Perm/Wash™ buffer at room temperature. Then, the cells were re–suspended with 100 μL BD Perm/Wash™ buffer plus antibody and incubated for 30 min at room temperature. Finally, the cells were washed with 1 ml washing buffer and re–suspended in 0.50 ml washing buffer. The cells were maintained at 4°C until analysed by flow cytometer.

After incubation with ALLO or vehicle, the BV-2 cells were detached and stained with annexin V coupled to PE and propidium iodide (PI), according to the manufacturer’s protocol (Invitrogen, Saint Aubin, France). The activation of caspase-3 was also investigated with the ‘PE Active Caspase-3 Apoptosis Kit’ (BD Biosciences). The cells were subsequently analyzed by flow cytometry to distinguish the different stages of cell death.

HM-1 cells were seeded in 6-well plates at a density of 3 × 10⁵ cells/well. After allowing the cells to adhere, drugs were added to each well for 24 hours. After washing, cells were stained using the PE Active Caspase-3 Apoptosis Kit (BD Biosciences) according to the manufacturer's protocol and analyzed using a flow cytometer system (BD Accuri C6). Each experiment was performed in triplicate and repeated at least three times.

SiHa cells were treated with 0–40 µM compounds 8–10 and 13–26. After 24 hrs, the cells were collected and assayed for apoptosis. Activated caspase-3 in SiHa cells was labeled with the phyco-erythrin-conjugated antibody in accordance with the manufacturer’s protocol (PE Active Caspase-3 Apoptosis Kit; BD Pharmingen, San Diego, CA) and analyzed by FACSCalibur flow cytometer (FL-2, FSC, BD Pharmingen). The assessment of apoptosis by flow cytometric technique was performed on SiHa cells that were plated in a 96-well plate as triplicate samples. The cancer cells were pretreated with various concentrations of compounds 8 and 10 for 0–6 hour after which the plate was centrifuged with a culture plate rotor (1000 rpm, 5 minutes). For analysis of nuclear fragmentation, propidium iodide (PI) buffer (0.3% Triton X-100, 40 mM Na-citrate, and 50 µg/ml PI) (Sigma) was added to the well plate. After 10 minutes of incubation period at room temperature, the plate was analyzed with LSRII flow cytometer equipped with HTS platform (PE-A channel). The fraction of sub-G0/G1 events was rated as a measure of apoptotic cell death.

MM cells were treated as indicated in 6-well plates seeded at a density of 1.0 × 10⁶ cells/well. Treated cells were harvested and processed with Cytofix/Cytoperm buffer before staining with PE-labeled active caspase-3 antibody (PE Active Caspase-3 Apoptosis kit, BD Pharmingen). Viable cells were identified using LIVE/DEAD Fixable Near-IR Dead Cell stain and activated caspase-3–positive cells were gated as an apoptotic population.