In This Issue
FEATURED NEW PRODUCTS
what it is
how it works
|NovaBright™ Secreted Placental Alkaline Phosphatase (SEAP) Enzyme Reporter Gene Chemiluminescent Detection Kit||400 assays||N10559|
|NovaBright™ Secreted Placental Alkaline Phosphatase (SEAP) Enzyme Reporter Gene Chemiluminescent Detection Kit||1,200 assays||N10560|
|NovaBright™ β-galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kit||200 assays||N10561|
|NovaBright™ β-galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kit||600 assays||N10562|
|NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Mammalian Cells||200 assays||N10563|
|NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Mammalian Cells||1,000 assays||N10564|
|NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Yeast Cells||200 assays||N10565|
|NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Yeast Cells||1,000 assays||N10566|
Click-iT® Biology Just Got Brighter — New Alexa Fluor® 555 Click-iT® EdU Cell Proliferation Assays and Detection Reagents
what it is
how it works
|Alexa Fluor® 555 azide, triethylammonium salt||0.5 mg||A20012|
|Alexa Fluor® 555 alkyne, triethylammonium salt||0.5 mg||A20013|
|Click-iT® EdU Alexa Fluor® 555 Imaging Kit *for 50 coverslips*||1 kit||C10338|
|Click-iT® EdU Alexa Fluor® 555 HCS Assay *2-plate size*||1 kit||C10352|
|Click-iT® EdU Alexa Fluor® 555 HCS Assay *10-plate size*||1 kit||C10353|
what it is
The Premo™ FUCCI cell cycle sensor enables live-cell imaging of cell cycle and division. As cells progress through the cell cycle, nuclear fluorescence changes from red to green.
what it offers
how it works
In 2008, Miyawaki and colleagues developed the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), a fluorescent protein–based sensor that employs red (RFP) and green (GFP) fluorescent proteins fused to different regulators of the cell cycle: Cdt1 and geminin. The temporal regulation of these proteins by ubiquitin results in the biphasic cycling through the cell cycle. Premo™ FUCCI Cell Cycle Sensor takes this technology one step further by using the BacMam gene delivery system—the prepackaged genetically encoded reagents are ready for immediate use. Simply add the Premo™ FUCCI reagents to your cells, treat with the BacMam enhancer, wash, incubate overnight for protein expression, and visualize cell cycle progression in populations of cells using fluorescence microscopy.
what it is
how it works
|Product||Species Reactivity*||Applications†||Quantity||Cat. No.|
|MEK1||Hu, Ms, Rt, X||WB, IP||100 µg||133500|
|MEK1 [pS298]||Hu, Ms, Rt||WB||10 blots||44460G|
|MEK2 [pT394]||Hu||WB||10 blots||44466G|
|MEK3/6 [pS189/pT193]/[207/211]||Hu||WB||10 blots||44470G|
|MEK4 [pS257/pT261]||Hu (Ms)||WB||10 blots||44474G|
|MEK7 [pS271/pT275]||Hu (Ms)||WB||10 blots||44478|
|p38 MAPK||Hu, Ms||WB||100 µg||AHO1202|
|p38 MAPK [pT180/pY182]||Hu, Rt (Ms, Cn, Mk)||WB, IHC, ICC||10 blots||44684G|
* Cn = canine, Hu = human, Mk = monkey, Ms = mouse, Rt = rat, X = Xenopus. ( ) = reactivity predicted but not tested.
Imaging Mitochondrial Calcium Sequestration — Organelle Lights™ Fluorescent Proteins Meet Classic Molecular Probes® Organelle Stains
The preferential accumulation of the calcium sensor rhod-2, AM in mitochondria has long been utilized to measure calcium flux, an important parameter given the vital role of mitochondria in maintaining intracellular calcium homeostasis. A limitation of using rhod-2 alone is that mitochondria are only visible after calcium uptake; however, determining mitochondrial location prior to the sequestration of calcium can provide important information about the positioning of individual mitochondria with respect to spatially localized intracellular calcium release. For more accurate measurement of mitochondrial calcium flux, the rhod-2 calcium indicator can be combined with probes for observing mitochondrial morphology, such as Organelle Lights™ Mitochondria GFP, for enhanced calcium imaging in individual mitochondria.
Organelle Lights™ reagents are ready-to-use fluorescent protein constructs fused with signal peptides for accurate and specific targeting to sub-cellular compartments and structures. Using the passive mitochondrial marker Organelle Lights™ Mito-GFP, heterogeneities in mitochondrial uptake and important mitochondrial dynamics such as fission, fusion, and motility can be observed before, during, and after the uptake of calcium into mitochondria. Pairing the green-fluorescent Organelle Lights™ Mito-GFP with the red-orange–fluorescent rhod-2, AM calcium sensor allows the spatial and temporal aspects of mitochondrial calcium sequestration to be analyzed with respect to individual mitochondrial dynamics within a given cell and across populations of cells.
|Imaging mitochondrial calcium levels and dynamics with rhod-2, AM and Organelle Lights™ Mito-GFP. (A) HeLa cells were labeled with Organelle Lights™ Mito-GFP and treated with 5 µM rhod-2, AM for 15 min at 37°C. (BI) The region outlined in A is enlarged to show individual mitochondria within a single cell. (BII–III) Calcium release from internal stores following application of 10 µM histamine. Mitochondria in close proximity to the calcium release from internal stores are shown by the increase in the orange-red fluorescence of rhod-2, AM. The arrow in BII denotes a mitochondrion that has impaired mitochondrial calcium uptake, a detail that would not have been revealed using rhod-2, AM alone. The asterisk marks a previously motile mitochondrion that appears to have stopped moving following calcium elevation. Organelle Lights™ Mito-GFP and rhod-2, AM were imaged using standard FITC and TRITC filters, respectively, on a DeltaVision® Core microscope with a 40x objective lens. |
|Organelle Lights™ Mito-GFP||1 kit||O36210|
|Rhod-2, AM dye||1 mg||R1244|
High-efficiency Transient Transduction of Human Embryonic Stem Cell–derived Neurons
High-efficiency transient transduction of human embryonic stem cell–derived neurons With baculoviral vectors.
Zeng J, Du J, Lin J et al. (2009) Mol Ther Epub ahead of print.
How can we transiently manipulate human embryonic stem cells to improve transplantation efficiency?
Human embryonic stem cells (hESCs) are an important source of cells for the generation of human neurons for basic research and therapeutic applications. Transplantation of hESC-derived neurons has the potential to repair damaged or diseased neurons, but efforts in this area have been limited by the poor survival and low functional recovery of grafted hESC-derived neurons. Genetic manipulation of hESC-derived cells prior to transplantation is one possible method of improving transplantation efficiency, but chromosomal integration of transgenes poses the risk of permanent expression of potentially harmful genes. Transient transduction is preferable for the expression of genes that are only beneficial during transplantation. To address this challenge, Zeng and colleagues developed a set of baculoviral vectors and demonstrate their use for successful transient transduction of hESC-derived neurons.
To develop a vector optimized for transient transduction of hESC-derived neurons, Zeng and colleagues investigated whether insect baculovirus–based vectors were suitable as a gene delivery system in these cells. They tested various eGFP expression cassettes in addition to the commercially available Organelle Lights™ ER-OFP and Mito-OFP baculoviral systems for transgene expression in hESC-derived neurons and in the brains of nude mice after transplantation.
Baculoviral transduction of hESC-derived neurons was highly efficient (up to 80%), and transgene expression was detected as early as 1 day post-transduction. Transgene expression was stable for at least 1 month in cultured human neurons, and co-transduction of baculoviral vectors with Organelle Lights™ fluorescent protein constructs (using a modified protocol) resulted in successful targeting of the fluorescent proteins concurrent with eGFP transgene expression. Finally, baculovirus-modified hESC-derived neurons were transplanted into the brains of nude mice, where transgene expression was detected for up to 4 weeks after injection. Baculoviral transduction did not appear to inhibit neuronal function, and there was no apparent immune response to the transplanted cells, suggesting that baculoviral vectors are promising tools for the development of more efficient neural transplantation systems.
View bibliography reference
- Learn More about Organelle Lights™ Reagents
|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.
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Live-cell imaging of cell cycle and division. U2OS cells were transduced with Premo™ FUCCI Cell Cycle Sensor, then imaged the next day using a 40x objective and standard FITC/TRITC filter sets. Time lapse imaging was performed on the DeltaVision® Core microscope. Images were collected every 15 min for 18 hr.
Watch the Video!
Epitope Tag Antibodies
Over the years, a number of different epitope tags have been made available to researchers studying protein expression. The tags are recognized by antibodies—which can in turn be visualized by standard secondary detection techniques—and have proven useful for detecting proteins of interest via western blotting, ELISA, immunostaining, and immunoprecipitation. Invitrogen offers antibodies against several of the most popular epitope tags, including V5, Xpress, myc, histidine (his), and hemagglutinin (HA). Each antibody is also available in several conjugate forms and includes complete protocols for use.
- Browse our complete offering of Epitope Tag Antibodies
Myc and V5 tags detected using epitope tag antibodies. (A) Myc tag detected with anti-myc antibody. (B) V5 tag detected with anti-V5 antibody.
|Anti-V5 Antibody||50 µL||R96025|
|Anti-Xpress™ Antibody||50 µL||R91025|
|Anti-myc Antibody||50 µL||R95025|
|Anti-histidine (c-terminal) Antibody||50 µL||R93025|
|Anti-HA Antibody||100 µg||326700|
|Environmental Sustainability Programs|
Invitrogen, part of Life Technologies, is dedicated to protecting the environment. We have ongoing environmental sustainability programs that deliver products to reduce your environmental footprint, while helping you achieve your research goals.
| New Web Resource for IDC Latex Microspheres |
Invitrogen offers a wide selection of UltraClean™ surfactant-free latex microspheres—also referred to as latex beads—for research and commercial applications. We have recently redesigned our IDC latex microspheres web page to include more information about the different surface modifications, detailed background information on the physical properties of microspheres, and a guide to selecting the best microsphere for your application. Also included are protocols for attaching latex microspheres to proteins or other biomolecules.
Post-Sorting Analysis Enabled by Low Toxicity of Cell Cycle Stain
Vybrant® DyeCycle™ Ruby Stain is used for cell cycle analysis in live cells by flow cytometry using the common 635 nm red laser for excitation. In collaboration with Derek Davies and Tina Luke from the FACS Laboratory at London Research Institute-Cancer Research UK, Invitrogen scientists have shown that Vybrant® DyeCycle™ Ruby Stain has low toxicity in live cells. This means that cells stained with Vybrant® DyeCycle™ Ruby Stain can be sorted on a flow cytometer into different phases of the cell cycle and then re-grown for subsequent analyses.
- Learn More about Vybrant® DyeCycle™ Ruby Stain
Post-sorting analysis of cell number and viability. HL-60 cells (5 x 106) were suspended in 10 mL of medium, 10 µL Vybrant® DyeCycle™ Ruby Stain was added, and the mixture was incubated for 15 min at 37°C. The cells were pelleted and resuspended in 1 mL PBS with 5 µL DAPI (200 µg/mL). (A) Live gating on DAPI negative cells followed by singlet gating based on area and height and sorted G0/G1 population (675/20 blue laser excitation and 660/20 red laser excitation). Mod FIT LT software was used to model the cell cycle, and the G0/G1 cells were then re-grown and evaluated for cell count (B) and viability (C).
|Vybrant® DyeCycle™ Ruby Stain||400 assays||V10273|
|Vybrant® DyeCycle™ Ruby Stain||100 assays||V10309|
Molecular Probes® The Handbook
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