Lipotoxicity is caused by intracellular lipid accumulation and is indicative of mitochondrial disfunction. Lipotoxicity accelerates the degenerative process of cellular senescence, influencing cancer development.

Cellular Senescence and Mitochondrial Disfunction Due to Lipid Accumulation

    • ① Lipid Droplet Detection
      • The Lipi-series kits can clearly identify lipid droplets with an extremely low level of background fluorescence and specificity better than that of BODIPY 493/503 or Nile Red.

        We observed lipid droplet generation in young and aged mice using mouse liver adipose tissue (frozen sections).

        Lipid Droplet Detection

        Lipi-series kits are available in four colors (blue, green, red, and deep-red) and can be used for multiple stain analysis as shown in the following papers.

        Sample Multiple Staining Reference
        Brown Adipocytes Co-stained with MitoBright Green (Mitochondria), Hoechst 33342 (Nuclear) and Lipi-Red Masako, O. et al., "Exogenous Cytokine-Free Differentiation of Human Pluripotent Stem Cells into Classical Brown Adipocytes", Cells., 2019, 8(4), 373.
        hMGEC Co-stained with tandem RFP-GFP-tagged LC3B (autophagosome /autophagolysosome) and Lipi-Blue Kim, S. et al., "Eicosapentaenoic acid (EPA) activates PPAR? signaling leading to cell cycle exit, lipid accumulation, and autophagy in human meibomian gland epithelial cells (hMGEC)", The Ocular Surface, 2020, 18(3), 427-437.
        AdMSCs Co-stained with UCP1 antibody (Alexa Fluor 488), DAPI (Nuclear) and Lipi-Red Yukimasa, T. et al., "Transcriptome analysis reveals brown adipogenic reprogramming in chemical compound-induced brown adipocytes converted from human dermal fibroblasts", Scientific Reports, 2021, 11, 5061.

        Products

        Function CAT. No. Product Name Size
        Lipid droplet imaging LD01 Lipi-Blue 10 nmol
        LD02 Lipi-Green 10 nmol
        LD03 Lipi-Red 100 nmol
        LD04 Lipi-Deep Red 10 nmol
        Lipid droplet quantification (plate reader, FCM) LD05 Lipid Droplet Assay Kit-Blue 1 set
        LD06 Lipid Droplet Assay Kit-Deep Red 1 set

    • ② Mitochondria Dynamics
      • Abnormalities in the morphology and dynamics of mitochondria have been associated with cellular senescence. This association has inspired a demand to be able to observe mitochondrial dynamics over a long period.

        Fortunately, MitoBright LT has been confirmed as being retained in mitochondria even after 4 days in HeLa cells.

        Mitochondria Dynamics

        Products

        Function CAT. No. Product Size
        Mitochondria imaging/ Long-term mitochondria visualization MT10 MitoBright LT Green 20 μl
        400 μl
        400 μl x 3
        MT11 MitoBright LT Red 20 μl
        400 μl
        400 μl x 3
        MT12 MitoBright LT Deep Red 20 μl
        400 μl
        400 μl x 3
        Mitochondria imaging/ Capable of co-stained with immunostaining MT15 MitoBright IM Red for Immunostaining 20 μl
        20 μl x 3

    • ③ Senescence Marker Detection
      • Cellular Senescence Detection Kit – SPiDER-βGal allows for detection of Senescence-Associated-β-Galactosidase (SA-β-gal) with high sensitivity and ease-of-use. SPiDER-βGal is a new reagent for detecting β-galactosidase, possessing high cell permeability and high retentivity inside cells. SA-β-gal can be detected specifically in either fixed or living cells by using Bafilomycin A1 to inhibit endogenous β-galactosidase activity. We used SPiDER-βGal to analyze the associations between cellular senescence, mitochondrial membrane potential, and cell cycle phase proportions. Doxorubicin (DOX) acts to inhibit cell proliferation during G2/M phases of the cell cycle and induces cellular senescence. After adding DOX to A549 cells, higher histogram peaks for the G2/M phase (Cell Cycle Assay Solution Blue and Deep Red), elevated senescence markers (Cellular Senescence Detection Kit – SPiDER-βGal), and changes in mitochondrial membrane potential (JC-1 MitoMP Detection Kit) were observed.

        Senescence Marker Detection

        Products

        Function CAT. No. Product Size
        SA-β-gal detection/ Tissue sample SG02 SPiDER-βGal 20 µg x 3
        SA-β-gal detection/ Fluorescence microscopy, FCM SG03 Cellular Senescence Detection Kit - SPiDER-βGal 10 Assays
        SA-β-gal detection/ Plate reader SG05 Cellular Senescence Plate Assay Kit - SPiDER-βGal 20 tests
        100 tests
        Mitochondrial membrane potential detection MT09 JC-1 MitoMP Detection Kit 1 set
        Cell cycle assay C548 Cell Cycle Assay Solution Deep Red 50 tests
        C549 Cell Cycle Assay Solution Blue 50 tests

    • ④ Monitoring Mitochondrial Membrane Potential
      • JC-1, TMRE, and TMRM are widely used to monitor mitochondrial membrane potential. However, these dyes have limitations, such as low photostability and poor retention after aldehyde fixation. These limitation result in experiments with poor reproducibility.

        MT-1 MitoMP Detection Kit has high photostability and remains unquenched even in cells that have been fixed with paraformaldehyde after staining. These features allow the MT-1 Kit to produce highly reproducible results.

        In addition, the Imaging Buffer included in this kit minimizes background fluorescence and maintains cell vitality while the assay is being performed.

        Monitoring Mitochondrial Membrane Potential

        Products

        Function CAT. No. Product Size
        Mitochondrial membrane potential detection MT09 JC-1 MitoMP Detection Kit 1 set
        Monitoring mitochondrial membrane potential MT13 MT-1 MitoMP Detection Kit 1 set

    • ⑤ Mitophagy Detection and Lysosome Detection
      • The Mitophagy Detection Kit does not require protein expression/transfection. Mitophagy can be easily detected by simply adding reagents.

        Mitophagy Detection and Lysosome Detection

        The association between cellular senescence and mitophagy can be confirmed using the Mitophagy Detection Kit. Arsun, B. at al., “Age-associated changes in human CD4+ T cells point to mitochondrial dysfunction consequent to impaired autophagy “, Aging (Albany NY), 2019, 11(21), 9234–9263.

        As well, in order to investigate the influence and involvement of mitochondria in lipofuscinogenesis, Dr. König et al. analyzed lipofuscin amounts as well as mitophagy in senescent cells. Jeannette, K. at al., “Mitochondrial contribution to lipofuscin formation “, Redox Biol., 2017, 11, 673–681.

        Many types of small fluorescent probes are used for monitoring lysosomes in living cells. The LysoPrime Green overcomes known problems with fluorescent lysosome probes, such as lack of specificity for lysosomes and staining dependent on the lysosomal pH. In addition, the high-retentivity of LysoPrime Green enables long-term imaging experiments.

        Mitophagy Detection and Lysosome Detection

        LysoPrime Green staining had retained inside the lysosome even during the long periods of stimulation required for autophagy and mitophagy induction.

        Products

        Function CAT. No. Product Size
        Lysosome detection L261 LysoPrime Green – High Specificity and pH Resistance 10 µl x 1
        Autophagy detection D677 DAPRed - Autophagy Detection 5 nmol x 1
        Mitophagy detection MD01 Mitophagy Detection Kit 1 set
        MT02 5 µg x 3

    • ⑥ Lipid Peroxide Detection
      • Liperfluo is a fluorescent probe that can directly detect intracellular lipid peroxide. Lipid peroxidation plays a critical role in the specific form of cell death known as ferroptosis. A great amount of research in recent years has focused on ferroptosis.

        Brent, S. et al., “Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease “, Cell, 2017, 171(2), 273.

        Valerian, K. al., “Oxidized Arachidonic/Adrenic Phosphatidylethanolamines Navigate Cells to Ferroptosis “, Nat. Chem. Biol., 2017, 13(1), 81.

        Lipid Peroxide Detection

        Products

        Function CAT. No. Product Size
        Lipid peroxide detection L248 Liperfluo 50 µg x 5
        Mitochondrial lipid peroxide detection/ Ferroptosis research M466 MitoPeDPP 5 µg x 3
        Intracellular Fe2+ detection/ Ferroptosis research F374 FerroOrange 24 µg x 1
        24 µg x 3
        Mitochondrial Fe2+ detection/ Ferroptosis research M489 Mito-FerroGreen 50 µg x 2
        GSSG/GSH quantification/ Ferroptosis research G257 GSSG/GSH Quantification Kit 200 tests
        Glutamate quantification/ Ferroptosis research G269 Glutamate Assay Kit-WST 100 tests

    • ⑦ Glucose Uptake Detection
      • Glucose Uptake Assay Kit-Green allows for highly sensitive detection of cellular glucose uptake via fluorescence microscopy, flow cytometry, or microplate assay.

        Unlike fluorescent dye 2-NBDG, which decreases in fluorescence intensity in water, this kit uses a fluorescent dye with superior brightness.

        Glucose Uptake Detection

        Products

        Function CAT. No. Product Size
        Glucose uptake detection UP02 Glucose Uptake Assay Kit-Green 1 set
        Glucose quantification G264 Glucose Assay Kit-WST 50 tests
        200 tests

    • ⑧ Total ROS Detection and Mitochondrial Superoxide Detection
      • DCFH-DA is widely used for ROS detection, but it has some limitations like weak fluorescence signals and high background. ROS Assay Kit -Highly Sensitive DCFH-DA- was developed to correct these limitations. The dye employed in the kit allows ROS detection with higher sensitivity than DCFH-DA.

        Total ROS Detection and Mitochondrial Superoxide Detection

        Mitochondrial Superoxide Detection

        MitoSOX™ Red has widely been used to detect mitochondrial superoxide. However, the emission wavelength is the common red, which overlaps with other MMP detection probes such as TMRE, and is therefore not applicable for simultaneous staining with these other mitochondrial markers in a single sample.

        mtSOX Deep Red overcomes these limitations. This dye emits deep red fluorescence; its fluorescence does not overlap with emission wavelengths that other red fluorescent markers use. Furthermore, the mtSOX Deep Red is better able to selectively detect superoxide, compared to MitoSOX™ Red.

        Total ROS Detection and Mitochondrial Superoxide Detection

        (Blue) Nuclear stain: Hoechst33342 (Ex: 405 nm, Em: 450–495 nm)
        (Green) Mitochondrial mass stain: MitoTracker™ Green FM (Ex: 488 nm, Em: 500–550 nm)
        (Red) Mitochondrial membrane potential stain: TMRE (Ex: 561 nm, Em: 560–620 nm)
        (Purple) Mitochondrial superoxide stain: mtSOX Deep Red (Ex: 633 nm, Em 640–700 nm)

        Products

        Function CAT. No. Product Size
        Total ROS detection R252 ROS Assay Kit -Highly Sensitive DCFH-DA- 100 tests set
        Mitochondria superoxide detection MT14 mtSOX Deep Red – Mitochondrial Superoxide Detection 100 nmol x 1
        100 nmol x 3
        Lipid peroxide detection L248 Liperfluo 50 µg x 5
        Nuclear staining H342 -Cellstain-Hoechst 33342 Solution 1 ml

    • ⑨ Cell Cycle Analysis
      • Propidium iodide (PI) is generally used for cell cycle analysis via flow cytometry. As opposed to the PI method, Cell Cycle Assay Solution Blue/Deep Red has better membrane permeability and uses a dye with high specificity for DNA. These differences make it possible to analyze the cell cycle just by adding the reagent to a cell suspension. Additionally, Cell Cycle Assay Solution Blue/Deep Red allows you to concurrently use the highly versatile 488 nm laser wavelength.

        Cell Cycle Analysis

        Products

        Function CAT. No. Product Size
        Cell cycle assay C548 Cell Cycle Assay Solution Deep Red 50 tests
        C549 Cell Cycle Assay Solution Blue 50 tests

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