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.
Methods for monitoring mitochondrial morphology, dynamics, and number are usually based on small fluorescent molecules or plasmid transfection techniques. The use of plasmids requires the target protein to be stably expressed, while small fluorescent molecules are widely used because they can simply be added to cells. Among commercially available small fluorescent molecules, those containing the chloromethyl moiety are commonly used. However, these dyes have some limitations, including short-term retention in cells, decreased fluorescence intensity in serum, and high background.
Dojindo’s MitoBright LT dyes overcome these limitations. MitoBright LT dyes are designed to exhibit mitochondria retention for long-term visualization. In addition, the MitoBright LT dyes show stronger fluorescence signals compared with other commercially available dyes that contain the chloromethyl moiety. The MitoBright LT dyes offer three different color options (Green, Red and Deep Red), and are provided as a ready-to-use DMSO solution. A working solution can easily be prepared in a single dilution step with growth medium or HBSS.
|High Intracellular Retentivity||Stained in serum-contained media||Detect by Several Equipments|
High Intracellular Retentivity
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.
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.
Mitochondrial membrane potential dependence of MitoBright LT Deep Red
Mitochondrial condition in the FCCP treated HeLa cells was compared with untreated cells using MitoBright LT Deep Red or existing reagents by flow cytometry.
The fluorescence intensity of TMRE decreased significantly when treated with FCCP, but in MitoBright LT Deep Red and an existing reagent (Company T), fluorescence intensity remained unchanged. MitoBright LT Deep Red retention may not depend on membrane potential.
Observation of Internal Structure of Mitochondria by Super-resolution Laser Microscopy (STED)
Cybrid cells with the causative mutation of mitochondrial disease were stained with MitoBright LT Deep Red and observed using super-resolution laser microscopy, confirming abnormalities in the cristae structure of mitochondria.
Control cybrid cells
Cybrid cells with mitochondrial
Dye：MitoBright LT Deep Red (100 nmol/l)
Instrument：Leica super-resolution laser microscopy TCS SP8 STED 3X
Ex. 640 nm / Em. 650-700 nm
STED laser：775 nm
1. Seed the cells in a glass base dish and incubate for 2 days (37oC, 5% CO2).
2. Remove the medium and add MitoBright LT Deep Red (100 nmol/l) prepared in L-15 medium (containing 10% FBS).
3. Incubate for 45 minutes (37oC, 5% CO2).
4. Remove the supernatant and wash twice with HBSS.
5. Add L-15 medium (containing 10% FBS) and observe with a super-resolution laser microscope (Leica TCS SP8 STED).
This data was kindly provided by Dr. I Osawa and Dr. Y. Fujita of the Research Team for Aging Control at the Tokyo Metropolitan Institute of Gerontology.
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.
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 Deep Red 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: 640 nm, Em: 650-700 nm
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.
Fixed cell imaging
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.)