Fam devd fmk

One- and two-dimensional cell cycle analyses, apoptosis determined by fluorochrome-labelled inhibitor of caspases (FLICA, FAM-DEVD-fmk, Immunochemistry Technologies) and immunofluorescence analyses by flow cytometry were performed according to manufacturers instructions or as described previously [11 (link)] and detailed in the electronic supplementary material.

BV-2 cells were cultured and prepared to detect by flow cytometric staining with propidium iodide (PI)-PerCP and caspase-1-FITC (FAM-YVAD-FMK, Immunochemistry Technologies) for NLRP3 inflammasome activation; PI-PerCP and caspase-3/7-FITC (FAM-DEVD-FMK, Immunochemistry Technologies), and 7aad-PerCP and Annexin-V-APC for apoptosis (BD Biosciences). Briefly, the cultured cells at 37 °C were stimulated by LPS (100 ng/mL) for 3.5 h, incubation with or without 3-MA (5 mM) for another 30 min, and then incubation with DS (0.3, 3, and 30 μM for apoptosis measurement; 30 μM for NLRP3 inflammasome activation measurement) for 1 h. After stimulating with nigericin (5 μM), the harvested cells were washed twice with ice-cold PBS, and stained with 7aad or PI for 5 min at room temperature, Annexin-V for 15 min at room temperature, or FAM-YVAD-FMK (caspase-1) and FAM-DEVD-FMK (caspase-3/7) for 1 h at 37 °C, respectively. The samples were acquired in a FACS BD verse cytometer (BD Biosciences), and data were analyzed using FlowJo software (Version 10.0; Three Star).

Fresh green oleaster leaves were collected from the region of Tlemcen, Algeria. Athymic nude mice were purchased from Charles River Laboratories (France). Human colorectal cancer cell lines (HCT116, HCT8) and normal colon epithelial CCD 841 CoN cell line were purchased from American Type Culture Collection (Rockville, MD). The HCT-116 p53^+/+ and p53^-/- cell lines were kindly provided by B. Vogelstein [20 (link)]. Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Lonza Verviers SPRL (Belgium). Fura-2 AM was purchased from Life Technologies (France). Cell Proliferation Kit I (MTT) was from Sigma-Aldrich. Anti-cytochrome c antibody (37BA11) was purchased from abcam (France). Anti-eIF2α (5324), anti-P-eIF2α (3579), anti-P53 (2524), anti-caspase-9 (9508), and anti-cleaved PARP (9541) antibodies were obtained from Cell Signaling (France). Annexin V-APC was purchased from Biolegend (France) and 7-amino-actinomycin D (7-AAD) from BD Biosciences (Belgium). FAM-DEVD-FMK was obtained from Immunochemistry Technologies (USA). Trypsin was purchased from Gibco (USA). Tetramethylrhodamine methyl ester perchlorate (TMRM) and MitoSOX Red were obtained from Molecular Probes (France). All other chemicals were purchased from Sigma (USA).

Four hours after AMF exposure, cells were incubated with Fluorochrome-Labeled Inhibitors of Caspase-1 (FLICA, FAM-YVAD-FMK, excitation: 488 nm) or Caspase-3 (FLICA, FAM-DEVD-FMK, excitation: 488 nm) reagent (Immunochemistry technologies, Bloomington, MN, USA) in accordance with the manufacturer’s instruction. Next, cells were washed and analyzed by confocal microscopy (LSM510, Zeiss). The counting of labeled cells was carried out by analyzing confocal microscopy images representing populations of 2000–3000 cells/experiment using Image J software.

Platelets (1–5 × 10⁶) were incubated with FITC-conjugated anti-CD41 (BD Phamingen, CA) (1:20) for 30 min at 37 °C. Isotype-matched antibodies were used to control the nonspecific binding of antibodies. Platelets were distinguished by specific binding of anti-CD41 and characteristic forward and side scattering. A minimum of 10,000 gated events were acquired using a FACScalibur flow cytometer (BD Bioscience, CA). Assessment of mitochondrial function was measured using the probe tetramethylrhodamine ethyl ester (TMRE, Fluka Analytical) (100 nM, 10 min) to label mitochondrial membrane potential (ΔΨm). Active caspase-1, caspase 3/7, and caspase-9 were determined using green FAM-YVAD_FMK, FAM-DEVD-FMK, and FAM-LEDH-FMK fluorescent inhibitors of caspases (FLICA), respectively, which irreversibly bind to activated caspases (Immunochemistry Technologies).