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5 protocols using mitospy red cmxros

1

Mitochondrial Dynamics in RAW264.7 Cells

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RAW264.7 cells were plated overnight on 20-mm glass-bottomed cell culture dishes. After treatment with EspC, the cells were incubated with the pre-warmed medium containing 250 nM of MitoSpy Red CMXRos (Biolegend) for 20 min. After the cells were washed with PBS, they were fixed and permeabilized in Immunol Staining Fix solution (Biyuntian) for 20 min and blocked with 3% bovine serum albumin (BSA) in PBS. The cells were washed with PBS and incubated with anti-cytochrome c and anti-Bax for 2 h at 37 °C in the Immunol staining primary antibody dilution buffer (Biyuntian). After the cells were washed with PBS, they were re-stained with the appropriate Alexa Fluor 488-conjugated secondary antibodies for 1 h at 37 °C and stained with 0.1 µg/mL DAPI for 10 min at 25 °C. The cells were analyzed using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) (63X; NA, 1.4) oil immersion lens (HC PL APO CS2; zoom, 2.5X; speed, 400 Hz; line average and resolution, 4). Images were acquired and processed using the LAS AF Lite software.
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2

Mitochondrial Membrane Potential and Mass

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Cells were stained with 20 nM MitoSpy Red CMXRos (Biolegend #424801) in media for mitochondrial membrane potential in live cells and 200 nM in fixed cells at 37 °C. Cells were stained with 50 nM MitoSpy Green FM (Biolegend #424806) for mitochondrial mass in live cells at 37 °C.
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3

IPEC-J2 Cell Viability and Mitochondrial Function

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The IPEC-J2 cell was a courtesy from Prof. Yin Yulong, Institute of Subtropical Agriculture, Chinese Academy of Sciences. H2O2 (Sinopharm Group, China), DMEM-F12 medium (Shanghai Yuanpei Biotechnology, China), and fluorescein isothiocyanate dextran 4 kDa (FD4) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Trypsin (Beyotime Biotechnology, China), phosphate-buffered saline (PBS) (Bozan Biotechnology, China), penicillin-streptomycin (Solabao Biotechnology, China), fetal bovine serum (Gemini, Australia), CCK-8 kit (Beyotime Biotechnology, China), MitoSpy™ Red CMXRos (Biolegend, USA), immunofluorescence fixative (Sevier Biotechnology, China), Triton-X 100 (Sigma-Aldrich, St. Louis, MO, USA), Glycine (Sinopharm Group, China), DAPI (Beyotime Biotechnology, China), Goat Anti-Mouse IgG Dylight 594 (Earthox, USA), Goat Anti-Mouse IgG Dylight 488 (Earthox, USA), mitochondrial division inhibitor (Mdivi-1) (Selleck, USA), AMPK inhibitor (Compound C, CC) (Selleck, USA), Lipofectamine RNAiMAX, and Lipofectamine 2000 were obtained from Invitrogen (Invitrogen, USA).
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4

Mitochondrial Membrane Potential Assay

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Mitochondrial mass in live cells treated with indicated reagents was monitored using the probe MitoSpy™ Red CMXRos (Biolegend) at final concentration of 100 nM, according to the manufacturer’s instructions, and analyzed by flow cytometry.
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5

Mitochondrial Function and Cytoskeleton Imaging

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After 24 h of culture, cells were exposed to HA-NP suspension or leachate for 1 and 4 hours. The cell layer was then washed once with warm PBS and the mitochondria specific tracking dye, MitoSpy™ Red CMXRos (250 nM, BioLegend), was added and cultures were incubated for 30 min at 37 °C. Cells were washed twice with PBS, fixed in 3.7% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Afterward, cells were permeabilised with 0.1% Triton-X100 (Sigma-Aldrich) for 15 min and cell cytoskeleton filamentous actin (F-actin) was stained with Alexa Fluor 488-conjugated phalloidin (1:100, Molecular Probes) for 30 min followed by nuclei staining with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL, BD Bioscience) for 10 min. Images of the stained cells were acquired using the Selena S digital imaging system (Logos Biosystems).
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