SK-Hep1 or Hep3B cells were seeded into 6-well plates at 5 × 105 cells/well overnight and then treated with different concentrations (0, 30, and 40 µM) of fluoxetine for 48 h. The changes of ΔΨm were investigated by 3,3′-Dihexyloxacarbocyanine Iodide (DiOC6) (Enzo Life Sciences, Farmingdale, NY, USA) staining. This process was followed by incubating cells with 4 µM DiOC6 working solution for 30 min at 37 °C. Then, the cells were resuspended in 500 µL PBS buffer and analyzed using flow cytometry [43 (link),44 (link)].
3 3 dihexyloxacarbocyanine iodide dioc6
Manufactured by Enzo Life Sciences
Sourced in United States, United Kingdom
3,3′-Dihexyloxacarbocyanine Iodide (DiOC6) is a fluorescent dye used for various biological applications. It is a lipophilic cationic dye that preferentially stains the mitochondria of cells. The dye exhibits green fluorescence upon binding to the mitochondrial membrane.
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Lab products found in correlation
Epinephrine hydrochloride, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Wortmannin, by Bio-Techne (1 mentions)
Recombinant human tpo, by R&D Systems (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Akts473, by Cell Signaling Technology (1 mentions)
Pik 75, by Cayman Chemical (1 mentions)
Gapdh sc 25778 antibody, by Santa Cruz Biotechnology (1 mentions)
Stat5, by Cell Signaling Technology (1 mentions)
Phospho ser pkc substrate, by Cell Signaling Technology (1 mentions)
Txb2 elisa kit, by Enzo Life Sciences (1 mentions)
Erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
Aktt308, by Cell Signaling Technology (1 mentions)
Gsk 3α β s21 9, by Cell Signaling Technology (1 mentions)
Stat5 y694, by Cell Signaling Technology (1 mentions)
Chrono lume reagent, by Chrono-Log (1 mentions)
4 protocols using 3 3 dihexyloxacarbocyanine iodide dioc6
1
Fluoxetine Alters Mitochondrial Membrane Potential
2
Platelet Agonist and Inhibitor Assay
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The platelet agonists used were: protease-activated receptor 1 (PAR-1)-activating peptide (SFLLRN-NH2; Bachem, Bubendorf, Switzerland), crosslinked collagen-related peptide (CRP-XL) from R. Farndale (Department of Biochemistry, University of Cambridge, UK), and fibrillar HORM collagen (type I) derived from equine tendon (Nycomed, Konstanz, Germany). The platelet inhibitors used were: ARC tetrasodium salt (R&D Systems, Abingdon, UK), ASA (Sigma-Aldrich, Poole, UK), and wortmannin (Tocris, Bristol, UK). The platelet primers used were: long-IGF-1 recombinant protein (receptor grade – AM001; Immunological and Biochemical Test Systems, Binzwangen, Germany), epinephrine hydrochloride (Sigma-Aldrich), and recombinant human TPO (R&D Systems). d -phenylalanylprolyl-arginyl chloromethyl ketone (PPACK) was from Calbiochem (Merck Chemicals, Watford, UK), and heparin was from Sigma-Aldrich. The commercial TxA2 ELISA kit and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) were from Enzo Life Sciences (Exeter, UK). All other reagents were from Sigma (Poole, UK), unless otherwise indicated.
3
Mitochondrial ROS and Membrane Potential Assay
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To measure mitochondrial ROS, cells were incubated with 5 μM MitoSOX (Invitrogen) at 37 °C for 30 min in the dark. Sample fluorescence was measured by flow cytometry (CytoFLEXTM). To detect mitochondrial membrane potential, cells were incubated with 20 nM 3,3-Dihexyloxacarbocyanine iodide (DiOC6) (Enzo, New York, NY, USA) for 15 min or 5 μg/mL Rhodamine 123 (Sigma-Aldrich) for 30 min at 37 °C in the dark and then analyzed by flow cytometry (CytoFLEXTM).
4
Signaling Pathways in Platelet Activation
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Cross-linked collagen-related peptide (CRP-XL) was from R. Farndale (Department of Biochemistry, University of Cambridge, UK). Fibrillar HORM collagen (type I) derived from equine tendon from Takeda (Linz, Austria). Recombinant murine thrombopoietin (TPO) was from PeproTech (London, UK). D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK) was from Calbiochem (Merck Chemicals, Watford, UK). 3,3'-Dihexyloxacarbocyanine iodide (DiOC6) and TxB2 ELISA kit were from Enzo Life Sciences (Exeter, UK). CHRONO-LUME® reagent was from CHRONO-LOG (Labmedics, Abingdon, UK). Akt S473 (#4060), Akt T308 (#13038), Akt (#9272), ERK1/2 T202/Y204 (#9101), GSK-3α/β S21/9 (#9331), JAK2 Y1007/1008 (#3771), JAK2 (#3230), p110α (#4249), Phospho-(Ser) PKC Substrate (#2261), STAT5/ Y694 (#4322), and STAT5/ (#9358) were from Cell Signaling Technology (New England Biolabs, Hitchin, UK). GAPDH (#sc-25778) antibody was from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, UK). PD184352, VX-702 and TGX-221 were from Bio-Techne (Abingdon, UK). PIK-75 and A66 were from Cayman Chemicals (Cambridge Bioscience, Cambridge, UK). Secondary antibodies for immunoblotting were from Jackson Immunoresearch (Stratech Scientific, Ely, UK). All other reagents were from Sigma (Poole, UK), unless otherwise indicated.
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