In This Issue

FEATURED NEW PRODUCTS

 
Light Up Autophagy — CellLight® Autophagy Reagents

 Good Things in Bigger Packages — Click-iT® EdU

 Fast, Efficient, and Convenient Antibody Labeling — APEX™ Alexa Fluor® 568 and Biotin-XX Labeling Kits

New Products for Cell &Tissue Analysis
 
Find Antibodies From Invitrogen with our new selection tools


NEW APPLICATIONS


 
Real-Time Detection of Dysfunctional Cell Cycling

FEATURED NEW PRODUCTS

CellLight® Autophagy Reagents

what they are
Autophagy—literally, “self eating”— is used by cells to cope with various cellular stresses. Our ready-to-use CellLight® targeted fluorescent proteins are used to label specific organelles that can be targeted for degradation. The combination of these probes with autophagy-specific markers, such as the Premo™ Autophagy Sensor, enables multicolor imaging of the cellular components interacting with autophagosomes during their formation and fusion with lysosomes.



what they offer

  • Efficient transduction of mammalian cells, including "difficult" cells such as primary and stem cells
  • Multiplex analysis with cellular stains, including other CellLight® reagents, antibodies, or MitoTracker® and LysoTracker® dyes
  • Simplicity—add, incubate, and image
how they work
Cellular labeling with CellLight® reagents employs BacMam technology, which uses a modified insect cell baculovirus coupled with a mammalian promoter as a vehicle to efficiently deliver and express genes in mammalian cells. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high cotransduction efficiency, enabling multiple BacMam reagents to be used in the same cell.

Imaging Autophagy

Imaging autophagy and various organelles with BacMam 2.0.


Read More +

A549 cells were transduced with Premo™ Autophagy Sensor LC3B-GFP and either CellLight® Mitochondria-RFP (A), CellLight® Golgi-RFP (B), CellLight® Lysosomes-GFP (C), or CellLight® ER-RFP (D). 24 hours after transduction, cells were treated with 50 mM chloroquine overnight to favor autophagosome formation. Nuclei were labeled with Hoechst 33342.

Click-iT® EdU

what it is
Click-iT® EdU provides a simplified method for detecting nascent DNA synthesis. EdU (5-ethynyl-2´-deoxyuridine), the click chemistry–based thymidine analog alternative to BrdU for measuring proliferating cells, is now available in a 5-gram size.



what it offers

  • Convenient size for in vivo applications
  • Extremely bright signal—no amplification required
  • Significantly simplified method for dual pulse labeling and detection

how it works
EdU is an alkyne-containing nucleoside analog of thymidine whose detection is based on a click reaction, a copper (I)–catalyzed reaction between an azide and an alkyne. The small size of the detection azide allows the use of mild conditions to access EdU incorporated into DNA. Even with tissue samples that normally require long incubations, the Click-IT® EdU assay is complete in less than 90 minutes, without the need for secondary detection or signal amplification.
 
Cell Proliferation Mammary Tissue
Effects of estrogen treatment on cell proliferation in mammary tissue, detected using Click-iT®
EdU and BrdU.


Read More +

Adolescent female rats were administered 200 mg estradiol over the course of 7 days. Following the hormone treatment, 5-bromo-2´-deoxyuridine (BrdU) was injected intraperitoneally, 160 µg/g
of body weight. 3 days later, an equivalent amount of 5-ethynyl-2´-deoxyuridine (EdU) was
injected 2 hr before sacrifice. EdU was detected with the Click-iT® EdU Alexa Fluor® 647 Imaging
Kit. BrdU was detected with an anti-BrdU antibody and visualized with an Alexa Fluor® 488 goat
anti-mouse secondary antibody. Apoptotic cells were identified with a cleaved capsase-3 antibody
visualized with Alexa Fluor® 594 goat anti-rabbit secondary antibody. Nuclei were counterstained
with Hoechst 33342.

APEX™ Alexa Fluor® 568 and Biotin-XX Labeling Kits

what they are
The APEX™ Antibody Labeling Kits provide a convenient means to directly attach a fluorescent or biotin molecule to very small amounts of IgG antibody (10–20 µg). Additional kits for Alexa Fluor® 568 and Biotin-XX antibody labeling are now available.




what they offer

  • Reliable—produces conjugates with a stable, covalent attachment of superior fluorescent or biotin labels
  • Convenient—requires as little as 10–20 µg of IgG antibody per labeling reaction, regardless of any purity limitations
  • Complete—includes everything required to perform 5 separate antibody labelings, typically in 2.5 hr (~15 min of hands-on time)

how they work
The APEX™ Antibody Labeling Kits use a solid-phase labeling technique that captures the IgG antibody on the resin inside the APEX™ antibody labeling tip. Contaminants, including stabilizing proteins and amine-containing buffers, are simply eluted through the tip. After applying the amine-reactive label, a fluorescent or biotin IgG conjugate is ready for use in any imaging or flow cytometry application in as little as 2.5 hours, with minimal hands-on time.

 
Labeling Tubulin
Labeling of α-tubulin in bovine pulmonary artery endothelial (BPAE) cells.

Read More +
Fixed and permeabilized BPAE cells were treated with Tubulin-α (Bovine), Mouse IgG1, detected with Goat Anti-Mouse IgG (H+L) secondary antibody, labeled with the APEX™ Biotin-XX Kit, and counterstained with Alexa Fluor® 488 Streptavidin. Nuclei were stained with blue-fluorescent DAPI.

NEW APPLICATIONS

Real-Time Detection of Dysfunctional Cell Cycling

The ability to visualize the different phases of the cell cycle is important in identifying physiologically and pathologically relevant aspects of mitosis. While traditionally this has been difficult to do, the Premo™ FUCCI Cell Cycle Sensor now enables you to follow progression through the cell cycle via a dynamic color change from red to green. 

Developed by Miyawaki and colleagues, the fluorescence ubiquitination cell cycle indicator (FUCCI) is a genetically encoded indicator that employs Red Fluorescent Protein (RFP) and Green Fluorescent Protein (GFP) fused to different regulators of the cell cycle: cdt1 and geminin. Life Technologies has incorporated the FUCCI genetic constructs into the powerful BacMam gene delivery system, creating a simple and efficient method for labeling cells and following their division.

Premo™ FUCCI allows you to accurately visualize the length of time spent in G1 vs. S/G2, and investigate polyploidy, a hallmark of cancer, resulting from extended G2/M followed by entrance into G1/S phase without undergoing mitosis.


 

Two-color cell cycle mapping using the Premo™ FUCCI Cell Cycle Sensor. 

DEPARTMENTS

Buzzworthy

In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons.

Dieterich DC, Hodas JJL, Gouzer G et al. (2010)  Nat Neurosci 13(7):897–905.

Because they are manufactured from (and degraded into) a single pool of unlabeled amino acids, distinguishing newly synthesized proteins from the existing proteins within a cell is no small task. In a recent study to visualize new proteins in neurons, Dieterich and colleagues grew rat hippocampal neurons in the presence of azide-bearing methionine surrogates. Proteins that had incorporated the modified amino acids were detected by incubating the cells in the presence of a copper catalyst, a fluorescent tag (an Alexa Fluor® 488–, Alexa Fluor® 568–, or Alexa Fluor® 647–conjugated secondary antibody), and the triazole ligand (a “click” chemistry reaction). Brief exposure to the labeling reagents gave rise to a fluorescent signal in the somata. A slightly longer exposure allowed visualization of newly synthesized proteins in dendrites. The authors further note that the method is effective in dissociated neurons and in brain slices. Their hope is to modify the technique to understand protein synthesis as it relates to learning and memory.


Primary human skeletal muscle cells
 
Primary human skeletal muscle cells (HSkM-S cells, Cascade Biologics®) were cultured in poly-D-lysine–coated glass-bottomed petri dishes and transduced with CellLight® ER-GFP and CellLight® Golgi-RFP in complete media. The following day, HSkM-S cells were loaded with 1 µg/mL Hoechst 33342 and 25 nM MitoTracker® Deep Red for 5 min at 37°C in DPBS. Cells were washed three times and imaged on a DeltaVision Core® microscope.




Product
Quantity Cat. No.

Human Skeletal Myoblasts1 vial (small size)A12555
Order Now
CellLight® ER-GFP *BacMam 2.0*1 mLC10590
Order Now
CellLight® Golgi-RFP *BacMam 2.0*
1 mL
C10593
Order Now
Hoechst 33342
10 mL
H3570
Order Now
MitoTracker® Deep Red FM *special packaging*
20 x 50 µg
M22426
Order Now
 


Detecting Autophagy – Make sure your LC3B antibody is up to the challenge

The LC3 protein plays a critical role in autophagy. Normally this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the phagophore and can be used as a general marker for autophagic membranes. Several commercially available LC3 antibodies do not appear to detect autophagosomes, even after chloroquine induction. For reliable autophagy detection, it is vital to use an anti-LC3B antibody.

These images show a comparison of several LC3B antibodies for autophagy, including our LC3B Antibody Kit for Autophagy. Cells were treated with either a DMSO control or 50 μM chloroquine to induce phagosome accumulation. Following fixation and permeabilization, cells were stained with the recommended amount of anti-LC3 antibody and visualized with green-fluorescent Alexa Fluor® 488 goat anti-rabbit IgG. Nuclei were stained with blue-fluorescent Hoechst 33342. The perinuclear green vesicular structures are autophagosomes.

 
Anti-LC3 Supplier Control 50 µM Chloroquine
Company A
   
Company B
   
Company C
   
Invitrogen

 

Product Quantity Cat. No.  
LC3B Antibody Kit for Autophagy
1 kitL10382Order Now
Hoechst 33342
10 mLH3570Order Now
Alexa Fluor® 488 Goat Anti-Rabbit0.5 mLA11008Order Now
Alexa Fluor® 555 Goat Anti-Rabbit
0.5 mLA21248Order Now
Alexa Fluor® 594 Goat Anti-Rabbit
0.5 mLA11012Order Now
Alexa Fluor® 647 Goat Anti-Rabbit
0.5 mLA21244Order Now
    




Easily study dynamic changes, colocalization, and signaling events in live cells with CellLight® reagents.


CellLight® reagents are ready-to-use fluorescent protein constructs targeted to specific subcellular structures. Simply add the reagent to your cells and incubate overnight, and you’re ready to image your cells. All CellLight® reagents are also compatible with fixed-cell staining and high-content analysis.








Explore the high-performing Molecular Probes® and Dynal® streptavidin products.

One click is all you need to explore the various streptavidin products from the providers of high-quality detection (Molecular Probes®) and isolation (Dynal®) products. Molecular Probes® detection products include Alexa Fluor® dyes, Qdot® nanocrystals, classic dyes, and enzyme-labeled conjugates of streptavidin. We also offer streptavidin phycoerythrin (also known as SA-RPE), the standard for flow cytometry, microarrays, and ELISA applications. From the isolation experts, review the web content detailing the widely used, highly cited, and versatile streptavidin-coupled Dynabeads®.




The Molecular Probes® Technology Network

The Molecular Probes® Technology Network was designed to connect individuals around the world who are interested in fluorescence imaging and related applications. You can use this space to view and upload protocols, and to share information about your work in general. You can also view protocols from other researchers.




Molecular Probes® The Handbook

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