Distinguish Artifacts from True Functional Changes
The use of fluorescent dyes, such as tetramethylrhodamine, methyl or ethyl ester (TMRM or TMRE) to report changes in mitochondrial membrane potential is well established. However, it is often important to determine if an apparent change in the signal from a potentiometric dye is due to a change in mitochondrial function or to an artifact resulting from a change in mitochondrial mass, shape, or movement. For more accurate measurement of mitochondrial membrane potential, these membrane potential–sensitive dyes can be combined with probes for mitochondrial morphology (such as Organelle Lights™ Mitochondria GFP) to measure changes in potential that are independent of mitochondrial mass or movement.

Ratiometric Imaging of Mitochondrial Membrane Potential
Ratiometric imaging of mitochondrial membrane potential can be achieved by first transducing cells with an Organelle Lights™ mitochondria-targeted reagent, and subsequently loading cells with a fluorescent dye that is readily sequestered by active mitochondria. For example, colocalization of green-fluorescent Organelle Lights™ Mito-GFP and red-fluorescent TMRM can be used to generate a pseudoratiometric measurement of mitochondrial membrane potential (see figure). This approach enables the observation of “flickers” in mitochondrial membrane potential that cannot be attributed to movement of mitochondria between focal planes or to a change in mitochondrial mass. A similar approach can be used to monitor mitochondrial calcium with the rhod-2 AM dye or mitochondrial superoxide production with MitoSOX™ Red indicator. 

• Learn More about Mitochondrial Probes.
• Learn More about Organelle Lights™ Reagents.

 

Click image for a larger view

Dynamic imaging of mitochondrial membrane potential. Mitochondria of HeLa cells were labeled with Organelle Lights™ Mitochondria GFP (Cat. no. O36210) (A1), and loaded with 50 nM TMRM (Cat. no. T668) for 10 minutes at 37°C (A2). Colocalization of TMRM and GFP can be clearly seen (A3), confirming the specific accumulation of TMRM in mitochondria. (B) Images were acquired at 5 second intervals for 90 seconds. Polarized mitochondria display both red and green fluorescence; those that have depolarized lose red TMRM fluorescence but retain GFP fluorescence and therefore appear green (B4, and quantified in C). Over time, mitochondrial membrane potential is lost in one mitochondrion (denoted by the arrow in B1) whereas surrounding mitochondria remain polarized. The recovery of membrane potential in this single mitochondrion can be seen in the subsequent image. GFP and TMRM were imaged using standard FITC and TRITC filters, respectively, on a DeltaVision® Core microscope with a 40x lens.

Click-iT® EdU Enables Rapid and Sensitive Assessment of Unscheduled DNA Synthesis

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU). Limsirichaikul, S. et al. (2009) Nucleic Acids Res 37:e31.

How can we rapidly and reliably assess nucleotide excision repair deficiencies?
Xeroderma pigmentosum (XP), a genetic disorder of the nucleotide excision repair (NER) system, predisposes its sufferers to photosensitivity and UV-induced skin damage. Diagnosis of XP typically entails determining the level of damage-induced, non–S-phase, gap-filling DNA repair activity (termed "unscheduled DNA synthesis", or UDS). While UDS can be sensitively and accurately assayed by monitoring the incorporation of ³H thymidine, this methodology is extremely time- and labor-intensive, and incurs the problems associated with handling radioactive materials. Immunofluorescence assays based on BrdU incorporation are substantially faster, but sensitivity is also greatly reduced as compared to ³H thymidine.

Methods
In this study, Limsirichaikul and colleagues compared these two assays to one based on incorporation of Click-iT® EdU, a reactive thymidine analog that enables fluorescence detection by conjugation to an azide-containing fluorophore ("click" chemistry).

Results
Using UVC-irradiated primary human fibroblasts and detection with Alexa Fluor® 488 azide, the group reported comparable sensitivity with Click-iT® EdU to that observed with conventional ³H thymidine autoradiography. Furthermore, the total time required to perform the EdU assay—about half a day—was dramatically less than that required for the ³H thymidine–based assay and even modestly faster than the BrdU-based assay. The Click-iT® EdU assay was also shown to be compatible with immunostaining and latex-bead labeling, allowing for the incorporation of internal controls that are crucial to rigorous lab testing. The group suggests that Click-iT® EdU–based assays could become the standard tool for the diagnosis of XP.

• View bibliography reference.

• Learn More about Click-iT® EdU and DNA Synthesis.

Product	Quantity	Cat. no.
Alexa Fluor® 594 goat anti-mouse IgG	0.5 mL	A11005
Alexa Fluor® 488 goat anti-mouse IgG	0.5 mL	A11001
Alexa Fluor® 594 goat anti-rabbit IgG	0.5 mL	A11012
Click-iT® EdU Alexa Fluor® 488 Imaging Kit	1 kit	C10083

Free online technical webinars You are invited to join us for a series of biweekly technical webinars from the comfort of your desk. The webinars will initially focus on imaging-related applications, but we welcome your feedback for additional topics throughout the course of the year. Upcoming topics will be announced each month via email. Presentations will last approximately 45 minutes, followed by 15 minutes for live Q&A. Webinars Date Time Labeling Molecules with Fluorescent Dyes May 26, 2009 10:00 a.m. PT Mitochondrial Biology June 9, 2009 10:00 a.m. PT

Missed our previous webinars? Find our recorded webinars here!