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.

Result:

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.

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 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

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.

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% CO 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.)

Product Comparison

Fluorescence Properties