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V10 analysis software

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FlowJo V10 is a data analysis software for flow cytometry. It provides tools for visualization, gating, and data analysis of flow cytometry experiments.

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Lab products found in correlation

Facsaria 2 sorter, by BD (4 mentions) Lsrfortessa, by BD (2 mentions) Facsdiva, by BD (2 mentions) Facsaria 2, by BD (2 mentions) Facsdiva software, by BD (2 mentions) Cellrox deep red reagent, by Thermo Fisher Scientific (2 mentions) Lsrii facs analyzer, by BD (1 mentions) Facsverse, by BD (1 mentions)

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FlowJo v10.2 single cell analysis software FlowJo Single Cell Analysis Software v10 FlowJo® V10 flow cytometry analysis software FlowJo software analysis

10 protocols using v10 analysis software

1

Mitochondrial TMRM Fluorescence Quantification

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Isolated mitochondria were supplemented with tetramethylrhodamine methyl ester (TMRM; Thermo Fisher Scientific, T668, 2nM in MSB) for 10min on ice before analysis on a BD LSRFortessa with BD FACSDiva software and violet 405nm, blue 488nm, yellow-green561nm and red 640nm lasers. Snap-Cell Oregon Green was detected in the GFP/FITC channel, Snap-Cell 647-SiR in the APC/A-647-channel, and TMRM in the PE-channel. FlowJo V10 analysis software (FlowJo LLC) was used. Plots are contour plots (5 % probability contouring threshold, with outliers). Median TMRM fluorescence intensity, normalised to the parent population, was analysed.
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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2

Mitochondrial age-class separation by FACS

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For separation of mitochondrial age-classes, mitochondrial fractions were sorted on a BD FACSAria II sorter (lasers: near UV 375nm, blue 488 nm, red 633nm; or violet 405nm, blue 488nm, yellow-green 561nm, red 633 nm) with a 70 μm nozzle (FSC 0.5× or 1.0× ND filter). Snap-Cell Oregon Green was detected in the GFP/FITC channel, and Snap-Cell 647-SiR in the APC/A-647-channel, using BD FACSDiva software versions 7 and 8. FlowJo V10 analysis software (FlowJo LLC) was used for data analysis. Plots shown are contour plots (5 % probability contouring threshold, with outliers).
Mitochondria were FACS-sorted and pelleted at 21,000g for 20 min. Supernatant was removed and mitochondrial pellets were snap-frozen in liquid nitrogen or on dry ice, or lysed for Western blotting.
To determine gating, separate samples of isolated mitochondria were incubated on ice for 30 min with MitoTracker Deep Red FM (40nM in MSB) before analysis on a BD FACSAria II sorter (lasers: blue 488nm, red 633nm, near UV 375nm), using the APC-channel.
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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3

HMEC Cell Sorting Workflow

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HMECs were synchronised and Snap-labelled as described above. Cells were collected and sorted on a BD FACSAria II or FACSAria fusion sorter (lasers: near UV 375nm, blue 488nm, red 633nm; or violet 405nm, blue 488nm, yellow-green 561nm, red 633nm) with an 85μm or 100μm nozzle (FSC 1.0x or 1.5x ND filter) using BD FACSDiva software (versions 7 and 8). Snap-Cell Oregon Green was detected in the GFP/FITC channel, Snap-Cell 647-SiR in the APC/A-647 channel. FlowJo V10 analysis software (FlowJo LLC) was used for further data analysis. Plots are presented as contour plots (5 % probability contouring threshold, with outliers).
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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4

Isolation and Immunostaining of Mouse Spinal Cord Nuclei

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Nuclei of mouse spinal cords were isolated as described above. After filtering of the homogenate, nuclei were fixed with methanol for 10 min on ice, followed by two washing steps with nuclei incubation buffer. For intranuclear staining, nuclei were permeabilized with nuclei incubation buffer supplemented with 0.5% Triton X-100 for 15 min on ice. After blocking with nucleus incubation buffer and 10% NDS, primary antibody was added for 1 hour at room temperature, followed by a washing step and subsequent incubation in secondary antibody for 30 min at room temperature. Immunofluorescence was acquired on an LSR II FACS analyzer (BD Biosciences). Data analysis was performed with the FlowJo v.10 analysis software (FlowJo LLC).
Rothammer N., Woo M.S., Bauer S., Binkle-Ladisch L., Di Liberto G., Egervari K., Wagner I., Haferkamp U., Pless O., Merkler D., Engler J.B, & Friese M.A. (2022). G9a dictates neuronal vulnerability to inflammatory stress via transcriptional control of ferroptosis. Science Advances, 8(31), eabm5500.
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5

Mitochondrial age-class separation by FACS

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For separation of mitochondrial age-classes, mitochondrial fractions were sorted on a BD FACSAria II sorter (lasers: near UV 375nm, blue 488 nm, red 633nm; or violet 405nm, blue 488nm, yellow-green 561nm, red 633 nm) with a 70 μm nozzle (FSC 0.5× or 1.0× ND filter). Snap-Cell Oregon Green was detected in the GFP/FITC channel, and Snap-Cell 647-SiR in the APC/A-647-channel, using BD FACSDiva software versions 7 and 8. FlowJo V10 analysis software (FlowJo LLC) was used for data analysis. Plots shown are contour plots (5 % probability contouring threshold, with outliers).
Mitochondria were FACS-sorted and pelleted at 21,000g for 20 min. Supernatant was removed and mitochondrial pellets were snap-frozen in liquid nitrogen or on dry ice, or lysed for Western blotting.
To determine gating, separate samples of isolated mitochondria were incubated on ice for 30 min with MitoTracker Deep Red FM (40nM in MSB) before analysis on a BD FACSAria II sorter (lasers: blue 488nm, red 633nm, near UV 375nm), using the APC-channel.
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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6

Mitochondrial TMRM Fluorescence Quantification

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Isolated mitochondria were supplemented with tetramethylrhodamine methyl ester (TMRM; Thermo Fisher Scientific, T668, 2nM in MSB) for 10min on ice before analysis on a BD LSRFortessa with BD FACSDiva software and violet 405nm, blue 488nm, yellow-green561nm and red 640nm lasers. Snap-Cell Oregon Green was detected in the GFP/FITC channel, Snap-Cell 647-SiR in the APC/A-647-channel, and TMRM in the PE-channel. FlowJo V10 analysis software (FlowJo LLC) was used. Plots are contour plots (5 % probability contouring threshold, with outliers). Median TMRM fluorescence intensity, normalised to the parent population, was analysed.
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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7

HMEC Cell Sorting Workflow

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HMECs were synchronised and Snap-labelled as described above. Cells were collected and sorted on a BD FACSAria II or FACSAria fusion sorter (lasers: near UV 375nm, blue 488nm, red 633nm; or violet 405nm, blue 488nm, yellow-green 561nm, red 633nm) with an 85μm or 100μm nozzle (FSC 1.0x or 1.5x ND filter) using BD FACSDiva software (versions 7 and 8). Snap-Cell Oregon Green was detected in the GFP/FITC channel, Snap-Cell 647-SiR in the APC/A-647 channel. FlowJo V10 analysis software (FlowJo LLC) was used for further data analysis. Plots are presented as contour plots (5 % probability contouring threshold, with outliers).
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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8

Annexin V-FITC/PI Apoptosis Assay

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Cell apoptosis was detected using an Annexin V-FITC/propidium iodide (PI) cell apoptosis kit (cat. no. KGA108-1; KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Briefly, cells reseeded at a density of 1×105 cells/well in a 6-well plate were exposed to different pressures. Subsequently, the cells were washed with ice-cold PBS, collected in a 1.5 ml Eppendorf tube, and treated with Annexin V-FITC/PI for 10 min at room temperature according to the manufacturer's protocol. The cells were then analyzed using a flow cytometer (BD FACSVerse; BD Biosciences). Data were analyzed using FlowJo V10 Analysis software (FlowJo LLC).
Shen J., Sun Y., Shen S., Luo X., Chen J, & Zhu L. (2019). Pressure suppresses hepatocellular glycogen synthesis through activating the p53/Pten pathway. Molecular Medicine Reports, 19(6), 5105-5114.
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9

Quantifying Cellular Oxidative Stress

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Synchronised and Snap-labelled cells were loaded with 2.5μM CellROX Deep Red Reagent (Thermo Fisher Scientific, C10422) for 30min at 37°C, washed, trypsinised, collected, and analysed on a BD FACSAria II sorter (lasers: Near UV 375nm, Blue 488nm, Red 633nm; or Violet 405nm, Blue 488nm, Yellow-Green 561nm, Red 633nm) with a 85μm or 100μm nozzle (FSC 1.0× or 1.5× ND filter). Instruments were equipped with BD FACSDiva software, and FlowJo V10 analysis software (FlowJo LLC) was used for data analysis. Snap-Cell Oregon Green (old mitochondria) was detected in the GFP/FITC channel, CellROX in the A-647/APC channel.
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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10

Quantifying Cellular Oxidative Stress

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Synchronised and Snap-labelled cells were loaded with 2.5μM CellROX Deep Red Reagent (Thermo Fisher Scientific, C10422) for 30min at 37°C, washed, trypsinised, collected, and analysed on a BD FACSAria II sorter (lasers: Near UV 375nm, Blue 488nm, Red 633nm; or Violet 405nm, Blue 488nm, Yellow-Green 561nm, Red 633nm) with a 85μm or 100μm nozzle (FSC 1.0× or 1.5× ND filter). Instruments were equipped with BD FACSDiva software, and FlowJo V10 analysis software (FlowJo LLC) was used for data analysis. Snap-Cell Oregon Green (old mitochondria) was detected in the GFP/FITC channel, CellROX in the A-647/APC channel.
Döhla J., Kuuluvainen E., Gebert N., Amaral A., Englund J.I., Gopalakrishnan S., Konovalova S., Nieminen A.I., Salminen E.S., Muñumer R.T., Ahlqvist K., Yang Y., Bui H., Otonkoski T., Käkelä R., Hietakangas V., Tyynismaa H., Ori A, & Katajisto P. (2022). Metabolic determination of cell fate through selective inheritance of mitochondria. Nature cell biology, 24(2), 148-154.
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