Ez ldh cytotoxicity assay kit

Antibodies against VEGF (A-20), MMP-9 (H-129), and β-actin (I-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In addition, antibodies against PI3K Class III (D4E2), phospho-PI3K Class III (Ser 249), AKT (5G3), phospho-AKT (Ser473), p38 (D13E1), phospho-p38 Thr180/Tyr182 (D3F9), ERK (p44/42), phospho-ERK (Thr202/Tyr204), phospho-p65 (Ser536) (93H1), p65 (C22B4), phospho-JNK (Thr183/Tyr185), JNK, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). EGM^TM-2 Endothelial Cell Growth Medium-2 BulletKit^TM was purchased from Lonza (Walkersville, MD, USA). The EZ-Cytox cell viability assay kit and EZ-LDH cytotoxicity assay kit were purchased from DoGenbio (Seoul, Korea). Recombinant human tumor necrosis factor (TNF)-α was purchased from Peprotech (Rocky Hill, NJ, USA). Mayer’s hematoxylin solution was purchased from Muto Pure Chemicals (Tokyo, Japan).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Phosphate-buffered saline (PBS) was purchased from Corning (Corning, NY, USA). IBMX, dexamethasone, insulin, and Oil Red O powder were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen (Carlsbad, CA, USA). Enhanced chemiluminescence solution (WestGlow Pico PLUS solA and solB) was purchased from Biomax Co., Ltd. (Seoul, Korea). Bradford protein assay reagent was purchased from Bio-Rad (Hercules, CA, USA). The EZ-LDH cytotoxicity assay kit was purchased from DoGenBio (ITSBIO, Seoul, Korea). Compound C was purchased from Sejin CI Co., Ltd. PPARγ, FAS, phospho-acetyl-CoA carboxylase (p-ACC), total ACC (t-ACC), phospho-AMPK (p-AMPK), total AMPK (t-AMPK), phospho-extracellular signal-regulated kinase (p-ERK), total ERK (t-ERK), phospho-c-Jun N-terminal kinase (p-JNK), total JNK (t-JNK), phospho-Akt (p-Akt), total Akt (t-Akt), phospho-p38 (p-p38), and total p38 (t-p38) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). C/EBPα, C/EBPβ, C/EBPδ, SREBP1c, β-actin, anti-rabbit, and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Isolated spleen cells from experimental animals were cultured in a medium for 24 h. The medium used was Roswell Park Memorial Institute (RPMI) 1640 medium (Welgene Inc., Daegu, Republic of Korea), which was supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) and 1% penicillin–streptomycin (PS) solution (Welgene, Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea). Subsequently, the spleen cells were used as effector cells, whereas YAC-1 cells (NK-sensitive cell line) were used as target cells. The ratio of effector cells to target cells was adjusted to 5:1. Simultaneously, broccoli extract samples were added at 2 mg/mL concentration. Then, incubation was continued for 4 h, and NK cell activity was evaluated by measuring lactate dehydrogenase (LDH) produced by the cells at 450 nm using an EZ-LDH cytotoxicity assay kit (Dogenbio, Seoul, Korea) to determine cytotoxicity [12 (link)].