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Cell Navigator® Mitochondrion Staining Kit *NIR Fluorescence*

Fluorescence images of HeLa cells stained with Cell Navigator® Mitochondrion Staining Kit *NIR Fluorescence* using fluorescence microscope with a Cy5 filter set. Live cells were stained with mitochondria dye MitoLite™ NIR (Red). After fixation, the cells were labeled with F-actin dye iFluor® 488-Phalloidin (Cat#23115, Green) and counterstained with Nuclear Blue™ DCS1 (Cat#17548, Blue).
Fluorescence images of HeLa cells stained with Cell Navigator® Mitochondrion Staining Kit *NIR Fluorescence* using fluorescence microscope with a Cy5 filter set. Live cells were stained with mitochondria dye MitoLite™ NIR (Red). After fixation, the cells were labeled with F-actin dye iFluor® 488-Phalloidin (Cat#23115, Green) and counterstained with Nuclear Blue™ DCS1 (Cat#17548, Blue).
Ordering information
Price ()
Catalog Number22670
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)658
Emission (nm)691
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
658
Emission (nm)
691
Mitochondria are membrane-enclosed organelles found in most eukaryotic cells. Mitochondria generate most of the cellular supply of ATP. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process. Our Cell Navigator® fluorescence imaging kits are a set of fluorescence imaging tools for labeling sub-cellular organelles such as membranes, lysosomes, mitochondria, nuclei, etc. The selective labeling of live cell compartments provides a powerful method for studying cellular events in a spatial and temporal context. This particular kit is designed to label mitochondria in live cells with near infrared (NIR) fluorescence. The kit uses our proprietary dye that selectively accumulates in mitochondria probably via the mitochondrial membrane potential gradient. The NIR fluorescent mitochondrial stain used in the kit is an ideal choice for imaging live cells and tissues where low background, and high signal-to-noise ratio is critical. The mitochondrial indicator, a hydrophobic compound, easily permeates intact live cells and trapped in mitochondria after it gets into cells. This fluorescent mitochondrial indicator is retained in mitochondria for a long time since it carries a cell-retaining group. This key feature significantly increases the staining efficiency. The kit can be readily adapted for many different types of fluorescence platforms, such as microplate assays, immunocytochemistry and flow cytometry. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components and can be used for both proliferating and non-proliferating cells.

Platform


Fluorescence microscope

ExcitationCy5 filter
EmissionCy5 filter
Recommended plateBlack wall/clear bottom

Components


Component A: MitoLite™ NIR1 vial (100 µL, 500X DMSO stock solution)
Component B: Live Cell Staining Buffer1 bottle (50 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells
  2. Add Mitolite™ NIR working solution
  3. Incubate at 37°C for 30 minutes to 2 hours
  4. Analyze the cells under fluorescence microscope at Ex/Em = 660/693 nm (Cy5 filter set)

Important notes
Thaw all the components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 20 µL of 500X Mitolite™ NIR (Component A) into 10 mL of Live Cell Staining Buffer (Component B) to make Mitolite™ NIR working solution. Protect from light. Note: 20 µL of 500X Mitolite™ NIR (Component A) is enough for one 96-well plate. The optimal concentration of the fluorescent mitochondrial indicator varies depending on the specific application. The staining conditions may be modified according to the particular cell type and the permeability of the cells or tissues to the probe.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

For adherent cells:

  1. Grow cells either in a 96-well black wall/clear bottom plate (100 µL/well/96-well plate) or on cover-slips inside a petri dish filled with the appropriate culture medium.

  2. When cells reach the desired confluence, add equal volume of Mitolite™ NIR working solution.

  3. Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes to 2 hours.

  4. Replace Mitolite™ NIR working solution with Hanks and 20 mM Hepes buffer (HH buffer) or buffer of your choice (e.g. the buffer with growth medium at 1:1 concentration).

  5. Observe the cells using a fluorescence microscope with Cy5 filter set (Ex/Em = 660/693 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained.

For suspension cells:

  1. Centrifuge the cells at 1000 rpm for 5 minutes to obtain a cell pellet and aspirate the supernatant.

  2. Resuspend the cell pellets gently in pre-warmed (37°C) growth medium, and add equal volume of Mitolite™ NIR working solution.

  3. Incubate the cells in a 37°C, 5% CO2 incubator for 30 minutes to 2 hours.

  4. Replace Mitolite™ NIR working solution with Hanks and 20 mM Hepes buffer (HH buffer) or buffer of your choice (e.g. the buffer with growth medium at 1:1 concentration).

  5. Observe the cells using a fluorescence microscope with Cy5 filter set (Ex/Em = 660/693 nm). Note: It is recommended to increase either the labeling concentration or the incubation time to allow the dye to accumulate if the cells do not appear to be sufficiently stained. Suspension cells may be attached to cover-slips that have been treated with BD Cell-Tak® (BD Biosciences) and stained as adherent cells.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)658
Emission (nm)691

Citations


View all 6 citations: Citation Explorer
Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering-induced Oma1 activation
Authors: Murata, Daisuke and Yamada, Tatsuya and Tokuyama, Takeshi and Arai, Kenta and Quir{\'o}s, Pedro M and L{\'o}pez-Ot{\'\i}n, Carlos and Iijima, Miho and Sesaki, Hiromi
Journal: The EMBO Journal (2020): e105074
Co-delivery of VP-16 and Bcl-2-targeted antisense on PEG-grafted oMWCNTs for synergistic in vitro anti-cancer effects in non-small and small cell lung cancer
Authors: Heger, Zbynek and Polanska, Hana and Krizkova, Sona and Balvan, Jan and Raudenska, Martina and Dostalova, Simona and Moulick, Amitava and Masarik, Michal and Adam, Vojtech
Journal: Colloids and Surfaces B: Biointerfaces (2017): 131--140
Inhibition of heme oxygenase-1 enhances the chemosensitivity of laryngeal squamous cell cancer Hep-2 cells to cisplatin
Authors: Lv, Xin and Song, Dong-mei and Niu, Ying-hao and Wang, Bao-shan
Journal: Apoptosis (2016): 489--501
Effective two-photon excited photodynamic therapy of xenograft tumors sensitized by water-soluble bis (arylidene) cycloalkanone photosensitizers
Authors: Zou, Qianli and Zhao, Hongyou and Zhao, Yuxia and Fang, Yanyan and Chen, Defu and Ren, Jie and Wang, Xiaopu and Wang, Ying and Gu, Ying and Wu, Feipeng
Journal: Journal of medicinal chemistry (2015): 7949--7958
Melatonin promotes adipogenesis and mitochondrial biogenesis in 3T3-L1 preadipocytes
Authors: Kato, Hisashi and Tanaka, Goki and Masuda, Shinya and Ogasawara, Junetsu and Sakurai, Takuya and Kizaki, Takako and Ohno, Hideki and Izawa, Tetsuya
Journal: Journal of Pineal Research (2015): 267--275

References


View all 70 references: Citation Explorer
Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detection
Authors: Feng J, Arriaga EA.
Journal: Electrophoresis (2008): 475
Calcium, mitochondria and apoptosis studied by fluorescence measurements
Authors: Roy SS, Hajnoczky G.
Journal: Methods (2008): 213
Fluorescence imaging of mitochondria in yeast
Authors: Swayne TC, Gay AC, Pon LA.
Journal: Methods Mol Biol (2007): 433
A fluorescence assay for peptide translocation into mitochondria
Authors: Martinez-Caballero S, Peixoto PM, Kinnally KW, Campo ML.
Journal: Anal Biochem (2007): 76
Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection
Authors: Duffy CF, MacCraith B, Diamond D, O'Kennedy R, Arriaga EA.
Journal: Lab Chip (2006): 1007
Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer
Authors: Elmore SP, Nishimura Y, Qian T, Herman B, Lemasters JJ.
Journal: Arch Biochem Biophys (2004): 145
A fluorescence-based technique for screening compounds that protect against damage to brain mitochondria
Authors: Kristian T, Fiskum G.
Journal: Brain Res Brain Res Protoc (2004): 176
Determination of the cardiolipin content of individual mitochondria by capillary electrophoresis with laser-induced fluorescence detection
Authors: Fuller KM, Duffy CF, Arriaga EA.
Journal: Electrophoresis (2002): 1571
Fluorescence imaging of metabolic responses in single mitochondria
Authors: Nakayama S, Sakuyama T, Mitaku S, Ohta Y.
Journal: Biochem Biophys Res Commun (2002): 23
Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy
Authors: Dedov VN, Cox GC, Roufogalis BD.
Journal: Micron (2001): 653