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Mitospy orange

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MitoSpy Orange is a fluorescent dye that selectively stains the mitochondria of live cells. It is a cationic, lipophilic dye that accumulates in the mitochondria due to their negative membrane potential. MitoSpy Orange can be used to visualize and monitor mitochondrial function in various cell types.

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Lab products found in correlation

Anti c myc, by Cell Signaling Technology (2 mentions) Foxp3 fixation permeabilization solution, by Thermo Fisher Scientific (2 mentions) Mitoview green mvg, by Biotium (2 mentions) Anti phospho p70s6k thr421 ser424, by Cell Signaling Technology (2 mentions) Lsrfortessa x 20, by BD (2 mentions) Sp8 confocal microscope, by Leica (1 mentions) Live dead aqua, by Thermo Fisher Scientific (1 mentions) Anti cd16 32 clone 93, by BioLegend (1 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

MitoSpy Orange CMTMRos (11 citations)

5 protocols using mitospy orange

1

Metabolic Profiling of CD4+ T Cells

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After culture, CD4+ T cells (0.2 × 106) were stained with metabolic probes diluted in T-cell culture media for 20 min at 37° celsius. Cells were then washed twice in staining buffer (PBS/2% FCS) and resuspended before analysis. Metabolic probes used included MitoSpy Orange (MSO) (25 nM; BioLegend, Cat# 424803), and MitoView Green (MVG) (50 nM; Biotium, Cat# 70054). For analysis of c-Myc expression and p70S6K phosphorylation, cells (0.2 × 106) were first fixed in FoxP3 fixation/permeabilization solution (eBioscience, Cat# 005523–00) for 20 min at 37° celsius, washed in FoxP3 permeabilization buffer and incubated with anti-c-Myc (Cell Signalling Technology, Cat# 5605T) or anti-phospho p70S6K (Thr421/ Ser424) (Cell Signalling Technology, Cat# 9204S) for 30 min at 4° celsius. After washing, cells were then incubated with secondary antibody anti-Rabbit IgG (H + L), AF555 (Invitrogen, Cat# A-21428) for 20 min at 4° celsius, followed by a final wash. Samples were run on the BD LSRFortessa X-20, data collected using BD FACSDiva Software, and analysed using FlowJo version 10.7.1 (BD Biosciences).
Bishop E.L., Gudgeon N.H., Mackie G.M., Chauss D., Roberts J., Tennant D.A., Maslowski K.M., Afzali B., Hewison M, & Dimeloe S. (2022). 1,25-Dihydroxyvitamin D3 suppresses CD4+ T-cell effector functionality by inhibition of glycolysis. Immunology, 166(3), 299-309.
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2

Metabolic Profiling of CD4+ T Cells

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After culture, CD4+ T cells (0.2 × 106) were stained with metabolic probes diluted in T-cell culture media for 20 min at 37° celsius. Cells were then washed twice in staining buffer (PBS/2% FCS) and resuspended before analysis. Metabolic probes used included MitoSpy Orange (MSO) (25 nM; BioLegend, Cat# 424803), and MitoView Green (MVG) (50 nM; Biotium, Cat# 70054). For analysis of c-Myc expression and p70S6K phosphorylation, cells (0.2 × 106) were first fixed in FoxP3 fixation/permeabilization solution (eBioscience, Cat# 005523–00) for 20 min at 37° celsius, washed in FoxP3 permeabilization buffer and incubated with anti-c-Myc (Cell Signalling Technology, Cat# 5605T) or anti-phospho p70S6K (Thr421/ Ser424) (Cell Signalling Technology, Cat# 9204S) for 30 min at 4° celsius. After washing, cells were then incubated with secondary antibody anti-Rabbit IgG (H + L), AF555 (Invitrogen, Cat# A-21428) for 20 min at 4° celsius, followed by a final wash. Samples were run on the BD LSRFortessa X-20, data collected using BD FACSDiva Software, and analysed using FlowJo version 10.7.1 (BD Biosciences).
Bishop E.L., Gudgeon N.H., Mackie G.M., Chauss D., Roberts J., Tennant D.A., Maslowski K.M., Afzali B., Hewison M, & Dimeloe S. (2022). 1,25-Dihydroxyvitamin D3 suppresses CD4+ T-cell effector functionality by inhibition of glycolysis. Immunology, 166(3), 299-309.
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3

Mitochondrial Length Quantification in TEPMs

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TEPMs were incubated in basal DMEM at pH 6.8 or 7.4 for 24 h. Cells were
washed and stained with 250 nM MitoSpy Orange (Biolegend) and 2 μg/ml
Hoechst (Sigma) for 20 min at 37°C. Cells were fixed with 4%
paraformaldehyde for 30 min at 4°C before they were washed, covered with
ProLong Diamond Antifade Mountant (Thermo Fisher), and sealed. Images were
acquired on a Leica SP8 Confocal microscope. Length measurements were taken in
Imaris 9.1.2 using the “Measurement Points” function. A minimum of
1000 lengths was measured by a “blinded” observer across
30–35 cells per experiment.
Jiang W., Le J., Wang P.Y., Cheng X., Smelkinson M., Dong W., Yang C., Chu Y., Hwang P.M., Munford R.S, & Lu M. (2021). Extracellular acidity reprograms macrophage metabolism and innate responsiveness. Journal of immunology (Baltimore, Md. : 1950), 206(12), 3021-3031.
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4

Multiparametric Flow Cytometry Analysis

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Cells were washed twice with PBS and stained using LIVE/DEAD Aqua (Invitrogen) according to the manufacturer’s instructions. Cells were washed two times with FACS buffer and Fc receptors blocked with anti-CD16/32 (clone 93; Biolegend). Cells were surface stained with the following antibodies: CD3 (Biolegend, 17A2, BV785 or PECy7), CD4 (Biolegend, GK1.5, BV605, APC, FITC or PE), CD8 (Biolegend, 53–6.7, BV421, APC/Fire 750), CD45 (Biolegend, 30-F11, PECy7), CD69 (Biolegend, H1.2F3, PECy7), PD1 (Biolegend, 29F.1.A12, BV421), LAG3 (Biolegend, C9B7W, PE). For intracellular and transcription factor staining cells were processed using a Foxp3/Transcription factor staining buffer set (eBioscience) and stained with antibodies, FoxP3 (eBioscience, FJK-16s, APC), Ki67 (Biolegend, 11F6, AF488), Glut1 (Abcam, EPR3915, AF488), TCF1/7 (Cell Signalling, 6790S, AF647). To enumerate antigen-specific T cells the PE-labeled dextramer (H-2Ld SPSYVYHQ) was used according to the manufacturer’s instructions. Mitochondrial membrane potential was determined using Mitospy-Orange (Biolegend) according to manufacturer’s instructions. Cell numbers were compared in tumors of individual mice at the same time-points after tumor cell inoculation; numbers were therefore normalized per gram of tumor tissue.
Lauder S.N., Smart K., Kersemans V., Allen D., Scott J., Pires A., Milutinovic S., Somerville M., Smart S., Kinchesh P., Lopez-Guadamillas E., Hughes E., Jones E., Scurr M., Godkin A., Friedman L.S., Vanhaesebroeck B, & Gallimore A. (2020). Enhanced antitumor immunity through sequential targeting of PI3Kδ and LAG3. Journal for Immunotherapy of Cancer, 8(2), e000693.
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5

Intracellular pH and Mitochondrial Staining

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The intracellular pH indicators pHrodo TM Green/Red AM were purchased from ThermoFisher Scientific (Waltham, MA), and MitoSpy Orange was purchased from Biolegend (San Francisco, CA). Cells were washed once with Live Cell Image Solution (LCIS) (Life Technoology/ThermoFisher Scientific, Grand Island, NY). Immediately prior to use, the pH indicator pHrodo TM Greenn/Red AM and the PowerLoad were mixed then diluted in the LCIS to produce working solution containing 0.5-1µΜ pH indicator and/or 100nM MitoSpy Orange.
The cells (1-2 x 10 7 /ml) were suspended in the working solution and incubated in a 37 o C water bath for 30 minutes. After the incubation, the cells were washed once in LCIS containing 1% FBS. The levels of staining were analyzed by flow cytometry.
Zeng W., Yang S., & Zhou B. (2021). Intracellular pH-regulated Cell Intrinsic Control of Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells.
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