In This Issue
FEATURED NEW PRODUCTS
what it is
how it works
what they are
how they work
|HCS CellMask™ Red stain||1 set||H32712|
|HCS CellMask™ Orange stain||1 set||H32713|
|HCS CellMask™ Green stain||1 set||H32714|
|HCS CellMask™ Blue stain||1 set||H32720|
|HCS CellMask™ Deep Red stain||1 set||H32721|
|HCS NuclearMask™ Blue stain||65 μL||H10325|
|HCS NuclearMask™ Red stain||125 μL||H10326|
what it is
how it works
what they are
how they work
Quantitate Mitochondrial Membrane Potential with Greater Accuracy — Organelle Lights™ Fluorescent Proteins Meet Classic Molecular Probes® Organelle Stains
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.
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.
|tetramethylrhodamine, methyl ester, perchlorate (TMRM)||25 mg||T668|
|tetramethylrhodamine, ethyl ester, perchlorate (TMRE)||25 mg||T669|
|Organelle Lights™ Mito-GFP||1 kit||O36210|
|Organelle Lights™ Mito-OFP||1 kit||O36222|
|Organelle Lights™ Mito-RFP||1 kit||O36229|
|rhod-2, AM dye||1 mg||R1244|
|MitoSOX™ Red mitochondrial superoxide indicator||10 x 50 µg||M36008|
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.
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).
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.
|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
|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.
Missed our previous webinars? Find our recorded webinars here!
|The juxtaposition of actin microfilaments and peroxisomes in subplasmalemmal regions visualized using Organelle Lights™ and Cellular Lights™ reagents. Human osteosarcoma cells were transduced with YFP targeted to peroxisomes (green), Cellular Lights™ Actin RFP (pink, Cat. no. C10127), and Cellular Lights™ Plasma Membrane CFP (blue, Cat. no. O36216) and imaged 14 hr later using CFP/YFP/RFP filters on a DeltaVision® Core microscope.|
|Cellular Lights™ Plasma membrane CFP||1 kit||O36216|
|Cellular Lights™ Actin RFP||1 kit||C10127|
Mitochondrial Stains—Visualizing Cellular Stress
Mitochondrial structure and function are key indicators of cellular stress, and mitochondrial defects have been implicated in several neurodegenerative diseases, including Alzheimer’s disease. Recent evidence suggests that the balance between mitochondrial fission and fusion is disrupted in response to several factors implicated in Alzheimer's disease. In addition, factors related to stroke, such as oxidative and nitrosative stress, and calcium dysregulation, can disrupt this balance. All of these effects may be mediated by a key motor protein surrounding mitochondria, dynamin-related protein-1 (Science 324:102 (2009)) or the GTPase mitofusin 2 (Curr Opin Cell Biol 18:453 (2006)) . These findings show that morphological visualization of mitochondria in live or fixed cells can be used as an effective model to understand inducers of these and other neurological pathologies.
Mitochondrial staining can be achieved using:
- Organelle Lights™ Reagents—fluorescent proteins targeted to mitochondria and delivered with efficient BacMam technology
- MitoTracker® Probes—dyes that accumulate in all mitochondria in live cells and are retained after fixation
- Potential-Sensitive Probes—dyes that only accumulate in healthy, hyperpolarized mitochondria, shifting their fluorescence properties with depolarization (e.g., JC-1 and JC-9)
- Primary Antibodies—extensive collection of antibodies targeting oxidative phosphorylation pathways and more
- Learn More about Mitochondrial Stains.
Live-cell imaging with Organelle Lights™ Endosomes-RFP and Mito-GFP in HeLa cells. HeLa cells were transduced with Organelle Lights™ Endosomes-RFP (Cat. no. O36231) and Organelle Lights™ Mito-RFP (Cat. no. O36229) and co-stained with 1 mg/mL Hoechst 33342 (Cat. no. H3570). Imaging was performed on live cells using a DeltaVision® Core microscope and standard DAPI/FITC/TRITC filter sets.
Less Sample, More Flexibility for Antibody Labeling
APEX antibody labeling kits—the newest addition to the Molecular Probes® antibody labeling kits family—offer a simple, convenient, and fast method of directly labeling as little as 10–20 µg of IgG antibody.
Visit www.invitrogen.com/apexlabeling to learn more.
Put Your Flow Cytometer’s Violet Laser to Work
Invitrogen is the recognized leader in providing world-class fluorescent reagents and antibody conjugates designed specifically for use on the violet laser. From immunophenotyping to dead-cell discrimination to cell cycle analysis, our portfolio unlocks the full multicolor potential of violet laser–equipped flow cytometers.
Visit www.invitrogen.com/violettools to learn more.
What your colleagues are saying about the Countess™ Automated Cell Counter
The Countess™ Automated Cell Counter uses trypan blue staining combined with a sophisticated image analysis algorithm to enable accurate cell and viability counts in just 30 seconds. The algorithm also measures the average size of live, dead, and total cells to give you all the data you need from your cell cultures without using a hemocytometer. Here’s what researchers are saying about how the Countess™ Automated Cell Counter has benefited their research:
“The Countess makes tissue culture work much easier and more productive. I wish I had one years ago!” –John McGrath, Dana-Farber Cancer Institute
“The Countess has worked out well for our lab. The Countess cell counts agree with our manual cell counts, and the Countess is much faster.” –Danielle Krebs, UBC Life Sciences Centre
“We found the Countess very helpful for our migration assays. Instead of spending hours counting cells to get results, we are able to quickly quantify our data! A definite time saver and well worth the cost!” –Holly, University of Rochester
“The Countess saves us hours of time during experimentally intense work days. We also appreciate the consistency of counts even with different users.” –Sarah, University of Illinois
Molecular Probes® The Handbook
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