Plot mean fluorescence of the viable cell population in the FL4 channel and normalize against that of untreated cells. Analyze the cells with an FC500 flow cytometer (Beckman Coulter, L’Hospitalet de Llobregat, Barcelona). Gently homogenize the cells and acquire at least 3000 cells in the FL4 channel. Resuspend the cells in 500 μL of 10 n M MTDR solution (Invitrogen, M22426) in DMEM in flow cytometry tubes and incubate for 15 min at 37☌ and 5% CO 2.
9.Ĭentrifuge at 770 × g for 5 min to eliminate the trypsin solution. 8.Īdd the cell suspension to a flow cytometry tube or leave it in the flow cytometry plate for plaque analysis. 7.Īdd 500 μL of complete DMEM and homogenize the cells by gentle pipetting. 5.Īdd 200 μL of trypsin (Gibco, 25300054) and incubate for 5 min or until the cells begin to detach from the well. 4.Īspirate the supernatant and transfer to flow cytometry tubes. CsA is used at 5 μ M and HCQ at 30 μg/mL. Incubate cells for 3 and 18 h at 37☌ and 5% CO 2 with 10 μ M CCCP, 10 μ M antimycin A, and 1 μ M oligomycin. Prepare the cocktail of AO in DMSO, CsA at 5 m M in DMSO, and HCQ at 30 mg/mL in water. Prepare the 25 m M stock CCCP solution (Sigma, C2759-100) in DMSO (Sigma, D8418). Seed cells (8 × 10 5) in a 24-well plate with 500 mL DMEM, high glucose (Biowest, L0104-500), supplemented with 220 m M glutamine (Sigma, G8540), 17 m M penicillin–streptomycin (Gibco, 15140122), and 10% fetal calf serum (complete DMEM). The detailed protocol is described later: 1. To confirm that a decrease in labeling is due to autophagic degradation, we block lysosomal degradation by treating cells with the classical lysosomal inhibitor hydroxychloroquine (HCQ) ( Boya et al., 2005) and prevent mitophagy using cyclosporine A (CsA), which blocks the mitochondrial permeability transition pore and has been previously used to prevent mitophagy by us and other research groups ( Carreira, Lee, Ghochani, Gustafsson, & Gottlieb, 2010 Domenech et al., 2015 Elmore, Qian, Grissom, & Lemasters, 2001 Kang & Hwang, 2009 Mauro-Lizcano et al., 2015 Rodriguez-Enriquez, Kim, Currin, & Lemasters, 2006). We treat cells of the SHSH-5Y human neuroblastoma cell line with these two mitophagy inducers for short (3 h) and long (18 h) periods and assess mitochondrial levels using the mitochondrion-selective probe MitoTracker Deep Red (MTDR). This can be achieved using the mitochondrial uncoupler CCCP (carbonyl cyanide m-chlorophenyl hydrazone), which reduces the mitochondrial membrane potential, or by blocking mitochondrial respiration with a combination of oligomycin and antimycin A (AO) ( Lazarou et al., 2015 Narendra, Tanaka, Suen, & Youle, 2008). Mitochondrial depolarization is the most common strategy used to induce mitophagy in vitro. Boya, in Methods in Enzymology, 2017 2 Mitophagy Evaluation in Cell Lines
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