Atp assay kit

MTT reagents and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO). The CellROX Green reagent, DMEM, FBS, trypsin, penicillin–streptomycin and Hanks Balanced Salt Solution were purchased from Thermo Fisher Scientific (Waltham, MA); Lipofectamine was obtained from Invitrogen (Carlsbad, CA). The Lowery assay reagent was from BioRad (Hercules, CA). The immunoblot PVDF (polyvinylidene difluoride) membrane and Immobilon reagent were both purchased from Millipore (St. Louis, MO). The Mitochondrial Membrane Potential kit and the ATP Assay kit were purchased from Cayman (Ann Arbor, MI). The Lactate Assay and Mitochondrial Isolation kits along with the PI/RNase solution were purchased from Abcam (Cambridge, MA). Anti-AMPK, p-AMPK (Thr172), FOXO3a, p-FOXO3a (Ser413) and MnSOD p-ATM, γ-H2AX, p-p53, Bim, Bid, Bax, cleaved-PARP, cleaved-caspases 3, 8, and 9, cytochrome C and Bcl-2 antibodies and the secondary anti-mouse IgG-horseradish peroxidase antibody were obtained from Cell Signaling (Danvers, MA).

The cells were treated with perifosine as described for the MTT assay. Quantification of cellular ATP was performed in cells cultured in 24-well plates using the ATP assay kit (Cayman Chemicals, Ann Arbor, Michigan, USA) as described earlier (53 (link)). The ATP concentration was subtracted from the calibration curve. The 3D models were disintegrated by trypsin and subjected to ATP assay using the same procedure.

Mulberroside C (Figure 1) was provided by ChemFaces (Wuhan, China). Mouse monoclonal to CD62P (P-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Fura 2-AM (2-acetoxymethyl) and Alexa Fluor 488-conjugated fibrinogen were obtained from Invitrogen (Eugene, OR, USA). The BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA). The serotonin ELISA kit was provided by Labor Diagnostika Nord GmbH and Co. (Nordhorn, Germany). Collagen, U46619, and thrombin were purchased from Chrono-Log Co. (Havertown, PA, USA). The thromboxane B₂ assay kit, cAMP, cGMP enzyme immunoassay kit, and ATP assay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Cell signaling (Beverly, MA, USA) supplied antiphospho-p38^MAPK, antiphospho-ERK (1/2), antiphospho-VASP (Ser¹⁵⁷), antiphospho-VASP (Ser²³⁹), antiphospho-cPLA₂ (Ser⁵⁰⁵), antiphospho-PI3K (Tyr⁴⁵⁸), antiphospho-Akt (Ser⁴⁷³), antiphospho-inositol-3-phosphate receptor type I (Ser¹⁷⁵⁶), antiphospho-PLC_γ2 (Tyr⁷⁵⁹), anti-β-actin, and antirabbit secondary antibodies.