05 Cell Staining

MitoBright LT Green

MitoBright LT Green

Mitochondrial Staining

  • High Intracellular Retention
  • Long-Term Visualization of Mitochondria
  • Provided as Ready-to-Use DMSO Solution
  • Allows for Collagen-coated Plates
  • Product code
    MT10  MitoBright LT Green
Unit size Price Item Code
20 ul $62.00 MT10-10
400 ul $123.00 MT10-12
400 ul x 3 $308.00 MT10-14

  Dojindo Laboratories' Long-term mitochondrial staining reagent MitoBright LT series is widely used around the world. Here, we present some of the referenced publications, especially the ones in red font, indicating that MitoBright LT series can long-term stain mitochondria for up to 5 days.

Product Name Samples Observation time
after staining
Instrument Publications
MitoBright LT Green Cell (U251) immediately Fluorescence microscope Pharmaceuticals
MitoBright LT Green Yeast cell (Lipomyces starkeyi) immediately Fluorescence microscope Genes to Cells
MitoBright LT Green Cell (HeLa) immediately Plate Reader Biomaterials
MitoBright LT Green Cell (Naive CD4+ T cells) immediately Fluorescence microscope Cell Reports
MitoBright LT Green Cell (CT26) immediately Flow Cytometer Journal of Radiation Research
MitoBright LT Green Cell (C2C12) 5 Days Fluorescence microscope Polymers
MitoBright LT Green Cell (BMM) 36 hrs or 3 Days Fluorescence microscope/
Flow Cytometer
JCI Insights
MitoBright LT Green Cells (mouse erythrocytes) immediately Fluorescence microscope Front. Cell. Infect. Microbiol.
MitoBright LT Red Cell (SH-SY5Y) immediately Fluorescence microscope Free Radical Biol. Med.
MitoBright LT Red Cell (MRC-5) immediately Fluorescence microscope The FEBS Journal
MitoBright LT Red Cell (A11 cells/ P29 cells) 3 Days Fluorescence microscope BMC Mol. Cell Biol.
MitoBright LT Deep Red Cell (HT-1080; MCF-10A; MCF-7; HCT-116 ) immediately Fluorescence microscope Small
MitoBright LT Deep Red Cell (HT-1080; MCF-10A; MCF-7 ) 8 hrs Fluorescence microscope Advanced Therapeutics
MitoBright LT Deep Red Cell (4T1) 24 hrs Fluorescence microscope Advanced Functional Materials
MitoBright LT Deep Red Cell (SW982) immediately Fluorescence microscope Gene
ORDER

Description

A mitochondrion is a multifunctional organelle not only involved in energy production in a cell, but other additional cellular functions. The active cycle of mitochondrial fusion and division induces morphological changes, which is called mitochondrial dynamics. Abnormalities in morphological control of mitochondria are associated with neurodegenerative diseases, metabolic disorders, aging, and so on. Therefore, the demand for long-term observation of mitochondrial dynamics has recently been increasing.

 

Manual

Technical info

HeLa cells were washed with HBSS (Hank’s Balanced Salt Solution) and subsequently stained with each of MitoBright LTs or an existing reagent. The culture medium was replaced with serum-containing medium, and mitochondria were observed after 4 days of incubation. As a result, fluorescence intensity of an existing reagent decreased significantly after 4 days, but in MitoBright LT, fluorescence intensity remained unchanged and mitochondria were clearly observable. Moreover, after further incubation, we confirmed that MitoBright LT was retained in mitochondria even after 7 days.

<Condition>
MitoBright LT Green 、(Company T)Green :Ex 488 nm/Em 500–560 nm
MitoBright LT Red 、(Company T)Red :Ex 561 nm/Em 560–620 nm
MitoBright LT Deep Red 、(Company T)Deep Red :Ex 640 nm/Em 650–700 nm

Stained in serum-contained media

Staining was performed with either MitoBright LT or an existing reagent in serum-containing or serum-free medium. In serum-containing medium, low fluorescence intensity was observed when using the existing reagent, however the cells stained with MitoBright LT experienced clear, resilient fluorescence of mitochondria

 

Detection by flow cytometry

Jurkat cells (3.2×105 cells/mL) were suspended in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, plated on a 35cm dish, and incubated overnight at 37˚C in a 5% CO2 atmosphere. RPMI medium was removed and replaced with 5mL of MitoBright LT working solution (0.1μmol/L). The cells were then incubated for 30 minutes at 37˚C. The working solution was removed and the cells were washed twice with 5mL of RPMI medium. Fresh RPMI medium was poured into the dish and cells were passaged and analyzed every 2 days by flow cytometry.

Detection of mitochondria using a collagen coated tissue plate.

Collagen-coated tissue culture plates are often used for examination of mitochondrial morphology because this imaging requires high magnifications.
The existing mitochondrial staining reagent bound to collagen, resulting with an increase in background staining, whereas the MitoBright LT series yielded clearly stained mitochondria.

Conditions:
Staining: HeLa cells were plated on a collagen coated tissue culture plate and cultured for 24 hours. The culture medium was removed, and the cells were washed with HBSS.
MitoBright LT Green working solution (100 nmol/L) was poured into the plate, the cells were incubated for 30 minutes. The solution was removed, and the cells were washed with HBSS. A fluorescence microscope was used for imaging of mitochondria.
Detection: Ex: 488 nm, Em: 500-560 nm

Result:
In mitochondria stained with an existing reagent, background staining was observed, but the mitochondria stained with MitoBright LT were clearly stained without having effect of background staining.

Fluorescence Properties

References

Open References

No. Sample Instrument Reference(Line)
1 Cell (U251) Microscope Y, Shinoda, K. Kujirai, K. Aoki, M. Morita, M. Masuda, L. Zhang, Z. Kaixin, A. Nomoto, T. Takahashi, Y. Tsuneoka, J. Akimoto, H. Kataoka, R. Rachi, A. Narumi, T. Yoshimura, S. Yano and Y. Fujiwara, ”Novel Photosensitizer β-Mannose-Conjugated Chlorin e6 as a Potent Anticancer Agent for Human Glioblastoma U251 Cells.", Pharmaceuticals., 2020, 13(10), 316.
2 Yeast (Lipomyces starkeyi) Microscope L. Duan and K. Okamoto, "Mitochondrial dynamics and degradation in the oleaginous yeast Lipomyces starkeyi", Genes Cells., 2021, 10.1111/gtc.12875 .
3 Cell (HeLa) Plate Reader W. Islam, Y. Matsumoto, J. Fang, A. Harada, T. Niidome, K. Ono, H. Tsutsuki, T. Sawa, T. Imamura, K. Sakurai, N. Fukumitsu, H. Yamamoto, H. Maeda., "Polymer-conjugated glucosamine complexed with boric acid shows tumor-selective accumulation and simultaneous inhibition of glycolysis ", Biomaterials., 2021, 269, (-), 120631.
4 Cell (Naive CD4+ T cells) Fluorescence microscope T. Kanno, T. Nakajima, Y. Kawashima, S. Yokoyama, H. K. Asou, S. Sasamoto, K. Hayashizaki, Y. Kinjo, O. Ohara, T. Nakayama and Y. Endo, "Acsbg1-dependent mitochondrial fitness is a metabolic checkpoint for tissue Treg cell homeostasis", 2021, doi:10.1016/j.celrep.2021.109921.
5 Cell (CT26) Flow Cytometer L. Sun, K. Morikawa, Y. Sogo and Y. Suiura, "MHY1485 enhances X-irradiation-induced apoptosis and senescence in tumor cells", 2021, doi:10.1093/jrr/rrab057.
6 Cell (C2C12) Fluorescence microscope C. Lin, P. Wu, K. Liu, Y. Fan and J. Yu, "An Environmental Friendly Tapioca Starch-Alginate Cultured Scaffold as Biomimetic Muscle Tissue", 2021, doi:10.3390/polym13172882.
7 Cell (BMM) Fluorescence microscope/
Flow Cytometer
W. Ling, K. Krager, K. K. Richardson, A. D. Warren, F. Ponte, N. Aykin-Burns, S. C. Manolagas, M. Almeida and H. Kim, "Mitochondrial Sirt3 contributes to the bone loss caused by aging or estrogen deficiency", 2021, doi:10.1172/jci.insight.146728.
8 Cells (mouse erythrocytes) Fluorescence microscope M. Niikura, T. Fukutomi, J. Mitobe and F. Kobayashi, "Roles and Cellular Localization of GBP2 and NAB2 During the Blood Stage of Malaria Parasites", 2021, doi:10.3389/fcimb.2021.737457.

 

 

Q & A

Q

Fixed cell imaging

A

1. HeLa cells were seeded on a μ-slide 8 well plate and cultured at 37°C overnight in a 5% CO2 incubator.
2. The supernatant was removed and the cells were washed with serum-containing medium twice.
3. 0.1 μmol/l MitoBright LT (Green/Red/Deep Red) working solution (serum-containing medium) was added and the cells were incubated at 37°C for 15-60 min in the 5% CO2 incubator.
4. The supernatant was removed and the cells were washed with PBS twice.
5. 4% paraformaldehyde (PFA) solution was added and the cells were fixed at room temperature for 15 minutes.
6. The supernatant was removed and the cells were washed with PBS twice.
7. The cells were observed under a fluorescence microscope.

Figure 1. Fluorescent images of 4% PFA fixed HeLa cells (Stain then Fix)


Note: Cells should be fixed with paraformaldehyde (PFA). Fixation with methanol or other solvents extracts lipids and results in poor staining.
Note: The staining has low tolerance for permeabilization after fixation, and cannot be used with detergent.
(Triton X-100, NP-40 etc.)

Handling and storage condition

Specification
Appearance: Yellow liquid
Absorbance: 0.600~0.800
Handling and storage condition
-20°C
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