Caspase glo 3 7 assay g8090

The lipids DC-Cholesterol/Dioleoyl Phosphatidylethanolamine (DOPE) (30:70, w/w), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl) amino] butylcarboxamido) ethyl]-3,4-di [oleyloxy]-benzamide (MVL5) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Patient tumor derived cells generated as described in [14 (link)] were a gift from Dr. Jeremy Rich at University of California, San Diego. FTH1 siRNA (Santa Cruz, Cat# 40575) and control silencer^™ firefly luciferase siRNA (Thermo Fisher, Cat #AM4629) were used for transfection. Antibodies used were as follows. FTH1 (Cell Signaling, Cat #4393, 1:500), phosphoϒH2AX (Cell Signaling Cat #9718, 1:500 western blot; 1:200 immunocytochemistry), β-actin (Sigma, Cat #A5411, 1:5000), EEA1 (Santa Cruz Cat# 137130, 1:200), TOMM20 (Santa Cruz Cat#17764 1:100 western blot, 1:250 immunocytochemistry) and Nestin (Abcam, Cat # 22035, 1:500). HRP-conjugated secondary antibodies for western blot were purchased from GE Healthcare. DiI, siGLO and Alexa Fluor antibodies were purchased from ThermoFisher. MTS (Cat#G3582) and Caspase-Glo® 3/7 assay (G8090) were purchased from Promega and LDH assay kit (Cat# 11644793001) from Sigma.

Individual ventricular specimen were rapidly cut into 4–6 pieces, briefly rinsed in 40 ml ice-cold PBS, immediately snap frozen in liquid nitrogen, and stored at -80°C. Assays for determination of small metabolic molecules (4-HAE, ATP, GSH/GSSG) were deproteinized prior to analysis using the perchlorate-based precipitation based Deproteinizing Sample Preparation Kit according to the supplier’s instruction (no. K808-200; BioVision). All assays were performed according to the manufacturer’s instructions with minor modifications: 4-HAE/MDA (Bioxytech LPO-586 kit; Oxis), 8-OHdG (Bioxytech 8-OHdG-EIA kit; Oxis), ATP (ENLITEN ATP Assay System Bioluminescence no. FF200; Promega), ADP (ADP-Glo Kinase Assay V6930; Promega), aconitase (Bioxytech Aconitase-340 kit; Oxis), caspase-3 and 7 (Caspase-Glo 3/7 Assay; G8090; Promega), caspase 8 (Caspase-Glo 8 Assay; G8200; Promega) catalase (Bioxytech Catalase-520 kit; Oxis), and GSH/GSSG (Bioxytech GSH/GSSG-412 kit; Oxis). Each sample was measured in duplicate. Final values were normalized by the total protein concentration for each sample determined prior to precipitation.