We used MitoTracker Red CMXRos (Thermo Fisher Scientific, catalog no. M7512) to measure the amount of active mitochondria (16 ) in BY4741 Δdtd strain expressing EcDTD (WT), EcDTD-(A102F), empty vector, and ScDTD (WT) constructs. Initially, primary cultures (3 ml each in SD media) were grown overnight. From these primary cultures, an equivalent of 0.2 OD600 cells were inoculated into a 5-ml synthetic glycerol medium with 100 μM copper for induction and grown at 30°C at 200 rpm until the OD600 reaches 1.0. As the cells are in the growth phase, 10 nM concentration of MitoTracker Red CMXRos was added to cells, and cells were allowed to grow further for 30 min. Afterward, the cells were pelleted, washed, and dissolved in 1 ml of water. These liquid cultures were then subjected to flow cytometry with excitation and emission of 579, and 599 nm, respectively, using a BD LSRFortessa Flow Cytometer instrument. For each culture, 10,000 events were recorded to estimate the MitoTracker Red CMXRos fluorescence.
Lsrfortessa flow cytometer instrument
Manufactured by BD
The BD LSRFortessa Flow Cytometer is a high-performance instrument used for multi-parameter analysis of cells and particles in suspension. It is designed to accurately measure and analyze the physical and fluorescent characteristics of these samples.
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Lab products found in correlation
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Anti cd3 bv650, by BD (1 mentions)
Diva software, by BD (1 mentions)
Fixable viability stain 510, by BD (1 mentions)
Anti cd8 percp cy5, by BD (1 mentions)
Intracellular staining reagents, by Thermo Fisher Scientific (1 mentions)
Anti tim 3 pe, by R&D Systems (1 mentions)
Anti hla dr pacificblue, by BioLegend (1 mentions)
Anti cd38 bv786, by BD (1 mentions)
Anti tigit apc, by Thermo Fisher Scientific (1 mentions)
Anti ki67 pe cy7, by BioLegend (1 mentions)
Anti cd4 af700, by BioLegend (1 mentions)
2 protocols using lsrfortessa flow cytometer instrument
1
Quantifying Mitochondrial Activity in Yeast
2
Phenotypic Profiling of Immune Cells
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Cryopreserved PBMCs were first stained with fixable viability stain 510 (BD Biosciences, San Diego, CA, USA) for 15 min to exclude dead cells, then washed, and surface stained with anti-CD3-BV650 (BD Biosciences), anti-CD4-AF700 (Biolegend, San Diego, CA, USA), anti-CD8-Percp-Cy5.5 (BD Biosciences), anti-CD38-BV786 (BD Biosciences), anti-HLA-DR-Pacific blue (Biolegend), anti-CD160-AF488 (eBioscience San Diego, CA, USA), anti-PD-1-BV605 (Biolegend), anti-TIGIT-APC (eBioscience), and anti-TIM-3-PE (R&D Systems, Inc., USA) at room temperature for 20 min. Cells were permeabilized and fixed with intracellular staining reagents according to the manufacturer’s instructions (eBioscience) before intracellular staining with anti-ki67-PE-Cy7 (Biolegend). Samples were then washed before acquisition and analysis on a BD LSRFortessa flow cytometer instrument with Diva software (BD Biosciences). Relative isotype controls or fluorescence minus one samples were prepared to facilitate gating. Data were analyzed using Flowjo Software version 10 (Tree Star Inc., Ashland, OR, USA).
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