Flow cytometry reagent

Eagle’s minimum essential medium (EMEM), fetal bovine serum (FBS), trypsin-0.05 % EDTA solution, and antibiotic solution were obtained from Gibco Laboratories (NY, USA). Phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺, pH 7.4, were supplied by PAA Laboratories GmbH, Gotzis, Austria. Trypan blue, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT), hydrogen peroxide, zinc aspartate, bovine serum albumin (BSA), and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich, Seelze, Germany. Flow cytometry reagents were obtained from BD Biosciences, San Jose, CA, USA. All salts and other cell culture solutions were of cell culture grade (Gibco Laboratories, NY, USA). Sterile and non-toxic plates, flasks, tips, and centrifuge tubes were obtained from Sarstedt, Numbrecht, Germany. Reagents for quantitative determination of zinc were from Merck, Darmstadt, Germany. Antioxidant and polyphenol analyses’ reagents were from Sigma-Aldrich, Seelze, Germany. Raw pure propolis was purchased from Apipol Farma, Myslenice, Malopolskie, Poland. In the present study, a representative mixture of propolis obtained from apiaries located in North Poland was evaluated [16 (link)].

M199 and RPMI 1640 media, fetal bovine serum (FBS) and antibiotic-antimycotic mix (100X) were purchased from GIBCO/BRL (Grand Island, NY, USA); 0.25% trypsin and tryple express were acquired from INVITROGEN (Waltham, MA, USA. Sterile plastic material for tissue culture was obtained from NUNC (Rochester, NY, USA) and CORNING (Glendale, AZ, USA). Flow cytometry reagents were purchased from Becton Dickinson (Franklin Lakes, NJ, USA). Tumor necrosis factor alpha (TNF-α) was purchased from R&D Systems (Minneapolis, MN, USA). Thymidine [methyl-³H] was supplied by Perkin Elmer (Boston, MA, USA). 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) was purchased from Molecular Probes, Invitrogen (Carlsbad, CA, USA). Peroxidase-labeled monoclonal antibody against Von Willebrand factor and all the fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies vs. human adhesion molecules were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TiO₂-NPs were acquired from Paris Drugstore (Mexico City, Mexico).

EA.hy926 (EAHY) endothelial cells were kindly provided by Professor Cora-Jean S. Edgell (Pathology Department, University of North Carolina, USA) and were previously described by Tseng et al. [40] . Human U937 mononuclear cells were obtained from American Type Culture Collection [41] . EAHY and U937 cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) and RPMI1640, respectively supplemented with 10% fetal bovine serum (FBS), antibiotics and glutamine (Sigma Chemical Company, USA). ELISA kits for PAI-1, uPA and suPAR were purchased from R&D Systems (Minneapolis, MN, USA). PGF_2α ELISA kits were obtained from Cayman Chemical (Ann Arbor, MI, USA). P-cresol and 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes (Invitrogen Detection Technologies, Grand Island, NY, USA). Flow cytometry reagents were from Becton Dickinson (Franklin Lakes, New Jersey, USA).

3,3'-Diindolylmethane (DIM) (catalog no. BML-GR207), p-AMPK inhibitor Compound C, intracellular Ca²⁺ chelator BAPTA-AM, SOCE nonspecific inhibitor 2-APB, Fluo3/AM obtained from Sigma-Aldrich (St. Louis, MO, USA), Mag-Fluo-4/AM (M14206) and lipofectamin 2000 were purchased from invitrogen (Carlsbad, CA, USA). DIM was dissolved in dimethylsulfoxide (DMSO) and prepared into 100mM concentration stock solution, which was stored at 4 °C. 3-[4,5-dimethylthizaol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT), Hoechst 33342, paraformaldehyde (PFA), bicinchoninic acid (BCA) protein assay kit (P0011) were purchased from beyondtime (Shanghai, China). The flow cytometry reagent was purchased from BD bioscience (San Jose, CA, USA). Primary antibodies against STIM1 (1:1000, abcam), phosptho-AMPK (1:1000, CST), phosptho-ACC (1:1000, CST), Calpain (1:1000, immunoway), phosptho-PERK (1:1000, CST), ATF6 (1:1000, CST), CHOP (1:1000, CST), phosptho-IRE1α (1:1000, abcam), Bcl-2 (1:1000, CST), Bax (1:1000, CST), cleaved-caspase3 (1:1000, CST), LC3B (1:1000, CST), primary antibodies against GAPDH (1:10000) and horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Dulbecco’s modified Eagle medium (DMEM) was purchased from Hyclone (MD, USA), primary antibodies anti-phosphory-p38 (1:1000), anti-cleaved caspase3 (1:1000), anti-cleaved PARP (1:1000), anti-Bax (1:1000), anti-p38 (1:1000), anti-caspase 3 (1:1000) were purchased from Cell Signaling Technology (MA, USA). Primary antibodies for PCNA and crystal violet dye were purchased from Bioss Biotechnology (Beijing, China). Primary antibody for β-actin and horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The flow cytometry reagent was purchased from BD Bioscience (San Jose, CA, USA). The p38 MAPK inhibitor SB203580, DIM (catalog no. BML-GR207), BAPTA-AM, A23187, Fluo-3/AM and Hoechst 33342 dye, were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 and DIM were dissolved in dimethyl sulfoxide (DMSO) as stock solution, respectively. The final concentration of DMSO did not exceed 0.1%.