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In This Issue
Fluorescence-Based Tools for Visualizing Autophagy — Premo™ Autophagy Sensors
||New Primary Anti-Mouse Qdot® Antibody Conjugate for Flow Cytometry — Mouse CD19 Qdot® 655 Conjugate
New Recombinant Proteins for Stem Cell Research — Recombinant Mouse LIF
See all of this month's New Products for Cell & Tissue Analysis
Tracking Proliferating Stem Cells With Click-iT® EdU
Buzzworthy — Loss of Autophagy in Erythroid Cells Leads to Defective Removal of Mitochondria and Severe Anemia In Vivo
- The View — Imaging Autophagy
Proven Performers — FIX & PERM® Cell Permeabilization Reagents
- On the Web — Alexa Fluor® Dyes, Autophagy
Check out the latest issue of BioProbes
FEATURED NEW PRODUCTS
what they are
LC3B plays a critical role in autophagy, and its localization can be used as a general marker for this vital process. Premo™ Autophagy Sensors enable the temporal and spatial visualization of LC3B.
what they offer
Real-time analysis—observe autophagy in live cells
Ease of use—one-step protocol
Flexibility—compatible with live- or fixed-cell analyses
how they work
Premo™ Autophagy Sensors combine the selectivity of an LC3B–fluorescent protein (LC3B-FP) chimera with the transduction efficiency of BacMam technology, facilitating unambiguous visualization of this protein with an easy one-step protocol. Simply add the BacMam LC3B-FP reagent to cells, incubate overnight for protein expression, then image and analyze. The kits include a LC3B-FP mutant, which serves to indicate whether punctate staining may be due to FP aggregation, and chloroquine, which causes accumulation of autophagosomes.
- Learn More about Autophagy
Detecting autophagy with the Premo™ Autophagy Sensor in live cells.
|Premo™ Autophagy Sensor LC3B-GFP
|Premo™ Autophagy Sensor LC3B-RFP||1 kit||P36236|
Rat anti–mouse CD19 antibody conjugated to Qdot® 655 nanocrystals, validated for flow cytometry.
what it offers
- More information with less sample, in less time
- Minimal single-laser compensation
Compatibility with existing organic dyes
Qdot® conjugated primary antibodies are designed for use on flow cytometers with a violet (405 nm) laser, such as the Attune™ Acoustic Focusing Cytometer. They increase the number of available dyes and provide greater flexibility when designing multicolor panels. Qdot® nanocrystals can be used in combination with existing organic dyes to increase the number of detectable parameters. They also provide the advantages of increased brightness and photostability.
See all of our Qdot® Antibody Conjugates
|Rat anti–Mouse CD19 Qdot® 655 Conjugate.
Read More +
Log fluorescence intensity profiles of mouse splenocytes gated on lymphocytes and analyzed on a BD LSR™ II flow cytometer using 405 nm excitation and the specified emission filters. The black line represents cells stained with Mouse CD19 Qdot® 655 Conjugate, and the gray line represents unstained cells.
what it is
The leukemia inhibitory factor (LIF) protein is most notable for its ability to prevent the differentiation of mouse embryonic stem cells (mESCs), enabling them to maintain pluripotency. LIF is also involved in a wide variety of processes, including inflammation, bone metabolism, development, and hematopoiesis.
what it offers
- High biological activity—more results with less protein
- High purity—no interference from other proteins or contaminants
- Proven compatibility—GIBCO® growth factors are bioassayed with GIBCO® media
how it works
Recombinant mouse LIF is available in a lyophilized format in 10 µg and 100 µg pack sizes. The biological activity of mouse LIF was measured via dose-dependent reduction of MTS within stimulated mouse M1 cells. In addition, inhibition of differentiation of mouse embryonic stem cells was observed using a concentration of 10 ng/mL of recombinant mouse LIF. The optimal concentration for each specific application should be determined by a dose response assay.
Dose-dependent reduction of MTS in LIF-treated mouse M1 cells.
Read More +
Mouse M1 cells were stimulated with GIBCO® Recombinant Mouse LIF and a competitor’s recombinant mouse LIF. Biological activity was measured via dose-dependent reduction of MTS. The ED50 of the GIBCO® recombinant mouse LIF was determined to be 2.99 pg/mL, while the ED50 of the competitor’s product was 10.86 pg/mL, which demonstrates higher bioactivity of GIBCO® recombinant mouse LIF.
|Recombinant Mouse LIF||10 µg||PMC4054|
|Recombinant Mouse LIF||100 µg||PMC4051|
The Click-iT® EdU assay is a superior alternative to traditional methods for detecting and quantitating newly synthesized DNA. Historically, active DNA synthesis has been measured by following the incorporation of the radioactive nucleoside [3H]-thymidine into DNA. This method was later replaced by immunodetection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® EdU assay also uses a nucleoside analog, but in this case it is detected not with an antibody but a click reaction—a copper-catalyzed covalent reaction between an azide and an alkyne. In the Click-iT® EdU assay, the EdU nucleoside contains the alkyne, and the fluorescent detection reagent contains the azide.
The small size of the detection reagent compared to an anti-BrdU antibody eliminates the need to denature the DNA for access to the incorporated nucleoside. Eliminating the harsh denaturation step—a step that often destroys antigen recognition sites and sample morphology—provides a method that is easier and more reliable to perform, even on delicate stem cells.
- Learn More about Click-iT® EdU Cell Proliferation Assays
- Learn More about other products for Stem Cell Research
||Labeling stem cell proliferation with Click-iT® EdU. Human mesenchymal stem cells were incubated with the nucleoside analog 5-ethynyl-2´-deoxyuridine (EdU). Following fixation and permeabilization, EdU was detected with a click reaction using the Click-iT® EdU Alexa Fluor® 594 Imaging Kit. Nuclei were counterstained with blue-fluorescent Hoechst 33342 before imaging with a DeltaVision® Core microscope using fluorescence and differential interference contrast (DIC).|
Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo.
Mortensen M et al. (2010) Proc Natl Acad Sci U S A 107:832–837.
Autophagy is critical for red blood cell differentiation
Removal of organelles is necessary for the differentiation of mammalian red blood cells from precursor cells. Mitochondria are known to be removed through the autophagy pathway. In this process, the cytoplasmic components targeted for removal are engulfed in a double-membraned vesicle (autophagosome), which then fuses with lysosomes for degradation of its contents. Atg7 encodes the E1-like enzyme required in both of the conjugation systems required for autophagosome formation.
Atg7 is a key player in erythrocyte development
To test the role of Atg7 in erythroid cells, Mortensen et al. produced a hematological Atg7 knockout mouse (organism-wide knockouts of autophagy genes are lethal). They observed that CFSE-stained erythrocytes, harvested from Atg7 knockout mice and injected into wild type mice, disappeared from the peripheral blood supply much more quickly than wild type erythrocytes injected at the same time. Also, these same peripheral blood erythrocytes were stained to a much higher degree with annexin V, indicating an increase in cell death. Taken together, these data suggest that an erythroid-intrinsic defect is responsible for the increased cell death observed.
Atg7-deficient erythrocytes accumulate damaged mitochondria
Over the course of development, erythrocytes from Atg7 knockout mice accumulated mitochondrion-derived superoxide (detected with MitoSOX™ Red Superoxide Indicator), and the mitochondria within the cells showed increasing depolarization (as judged by tetramethylrhodamine, methyl ester, perchlorate (TMRM)), which indicated that erythroid cells from Atg7 knockout mice were accumulating damaged mitochondria. The authors further showed that damaged mitochondria accumulated in the erythroid lineage but not in the myeloid lineage of the same animals. These data suggest that the removal of defective mitochondria is an essential step during erythropoiesis, and that Atg7 is a key molecular player in this process.
View the bibliography reference
- Learn More about Products for Mitochondria
|CellTrace™ CFSE Cell Proliferation Kit||1 kit||C34554|
|MitoSOX™ Red Mitochondrial Superoxide Indicator||10 x 50 µg||M36008|
|Tetramethylrhodamine, methyl ester, perchlorate (TMRM)||25 mg||T668|
Imaging autophagy with the Premo™ Autophagy Sensor, Organelle Lights™ reagents, and MitoTracker® dye. A549 cells were transduced with the Premo™ Autophagy Sensor LC3B-GFP (green) and Organelle Lights™ Golgi-RFP (red), and then treated with 50 µM chloroquine. Twenty-four hours later, cells were loaded with 50 nM MitoTracker™ Deep Red Dye (cyan) for 5 min at 37°C and washed in dye-free DPBS. Nuclei were labeled with Hoechst 33342 (blue). Cells were imaged on a DeltaVision® Core microscope with DAPI/FITC/TRITC/Cy5 filter sets.
FIX & PERM® Cell Permeabilization Reagents: Great Data Require Great Starting Material
To accurately analyze intracellular markers by flow cytometry, cells must be fixed and permeabilized to enable detection antibodies to reach intracellular proteins. Preparation of cells for these protocols requires a delicate balance of adequately permeabilizing the cells to allow antibody access while maintaining the overall morphology of the cells. The extensively referenced FIX & PERM® Cell Permeabilization Reagents are the recognized gold standard for fixing and permeabilizing cells prior to analysis by flow cytometry.
Benefits of the reagents include:
- Superior access to intracellular structures
- No alteration of morphological scatter
- Manufactured under GMP standards for reliable performance
- Fast, easy protocols with consistent results
- Well-referenced standard for intracellular staining
The FIX & PERM® Cell Permeabilization Reagents allow mild fixation and permeabilization of cells, leaving their morphological scatter characteristics intact. This flexibility enables accurate identification of previously undetectable intracellular markers such as cytoplasmic or nuclear enzymes, phosphoproteins, oncoproteins, cytokines, and immunoglobulins.
Numerous references confirm many technical advantages of these reagents:
|Allow simultaneous addition of permeabilization medium and fluorochrome-labeled antibodies|
|Eliminate the need for additional lysing solutions|
|Compatible with analysis of most cellular antigens|
|Reduce background staining|
|Perfect for use with Invitrogen™ LIVE/DEAD® Fixable Dead Cell Stains for dead-cell discrimination|
FIX & PERM® reagents are suitable for flow cytometry analysis of normal and malignant leukocyte populations derived from various human biological samples (e.g., blood, bone marrow, etc.).
- Learn More about FIX & PERM® Cell Permeabilization Reagents
|FIX & PERM® Fixation and Permeabilization Reagents
||4 x 5 mL, 200 tests||GAS004|
|Alexa Fluor® Dyes
It’s now easier than ever to find information about Alexa Fluor® dyes—the leading and most trusted fluorescent dyes available today. With our newly redesigned web pages, you can quickly navigate to product information, performance data, and comparisons to other dyes.
|New Autophagy Web Resource
Autophagy—literally, “self-eating”—is an essential cellular process that involves degradation of cellular components or entire cells. In normal cells, it helps regulate such fundamental processes as cell growth and homeostasis, but autophagy has also been implicated in pathological processes. Our new web resource makes it easy to find tools for autophagy research—including the first commercially available reagent for autophagy research in live cells, as well as a validated antibody kit.
Molecular Probes® The Handbook
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