Cellular Senescence and Mitochondrial Disfunction Due to Lipid Accumulation

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

• • 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).

      

      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.

      

      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.

      

      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.

      

      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.

      

      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.

      

      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.

      

      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.

      

      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.

      

      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.

      

      (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.

      

      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