ProbesOnline December 2009

ProbesOnline Special Edition: Another chance to see the most popular products and technologies featured in ProbesOnline during 2009.


The Next Generation of Antibodies ABfinity™ Recombinant Monoclonal Antibodies

Still Using BrdU? New and Improved Click-iT® EdU Cell Proliferation Assays

Illuminate Internalization Pathways in Real Time pHrodo™ Dextran for Endocytosis

Cell Proliferation and Cytotoxicity Assays CyQUANT® Direct Cell Proliferation Assay

Live Cell Cycle Analysis With Limited Cytotoxicity Vybrant® DyeCycle™ Ruby Stain

NEW Cell & Tissue Analysis Products

New Products for Cell &Tissue Analysis
See all of this month's New Products for Cell & Tissue Analysis

Visualize Tight Junctions and the Cytoskeleton Alexa Fluor® 488 Dye–Conjugated Primary Antibodies

Live-Cell Imaging and Fixed-Cell Analyses Organelle Lights™ Reagents

Live-Cell Imaging of Cell Cycle and Division Premo™ FUCCI Cell Cycle Sensor

Take Charge of Your Cell Counting Count Blood Cells With the Countess™ Automated Cell Counter

Optimize Cell Viability Assays for Multicolor Panels LIVE/DEAD® Fixable Dead Cell Stain Sampler Kit

Check out the latest issue of BioProbes


what they are
ABfinity™ antibodies are the next generation of antibodies, presented exclusively by Invitrogen. These antibodies are generated by cloning the specific antibody genes and producing them in a mammalian expression system. ABfinity™ technology provides the most specific antibodies available, bringing you highly reproducible results.

what they offer

  • Unmatched lot-to-lot consistency
  • High sensitivity and specificity
  • Extensive validation and characterization

how they work
Generating antibodies by expressing cloned antibody genes ensures that every lot of ABfinity™ antibodies produces the same results. These antibodies offer maximal sensitivity and specificity, allowing you to detect small amounts of only your intended targets. ABfinity™ antibodies are also extensively validated and characterized, giving you the confidence you need, right out of the tube—no further optimization is required.

 ABfinity™ rabbit momoclonal antibody

HeLa cells labeled with ABfinity™ rabbit monoclonal antibody against the eukaryotic initiation factor 4EBP1 [pT37] in the absence of peptides (left), and in the presence of a phosphopeptide (center) or nonphosphopeptide (right) used as an immunogen. Detection was performed with Alexa Fluor® 488 goat anti–rabbit IgG. Actin was stained with Alexa Fluor® 568 phalloidin.
Reactivity: validated (expected) Applications Quantity Cat. No.
PKC-θ [pT538] Hu (X, Rt, Ms, Cp, B) WB, F, IHC, IF/ICC 100 μg 700043
Rab11 Hu (X, Rt, P, Ms, Eq, Ch, B) WB, IF/ICC 100 μg 700184
Mnk1 [pT197/pT202] Hu (Z, X, Sw, Rt, P, Ms, Mk, Eq, Cp, Ch, Cn, B) WB, F, IF/ICC 100 μg 700242
Reactivity: B, bovine; Ch, chicken; Cn, canine; Cp, chimpanzee; Eq, equine; Hu, human; Mk, monkey (rhesus); Ms, mouse; P, primate; Rt, rat; Sw, swine; X, Xenopus; Z, zebrafish
F, flow cytometry; ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; WB, western blotting
what it is
The Click-iT® EdU assay is a superior alternative to traditional methods for detecting and quantitating newly synthesized DNA. The Click-iT® EdU imaging and HCS kits now include a 1X ready-to-go reaction buffer—eliminating stock solution preparations and careful additions of reagents. Also included is a new Click-iT® reaction rinse buffer that eliminates any foaming caused by the previous BSA-based wash buffer for crisp imagery and better data.

what it offers

  • Rapid detection—results in only 30 minutes
  • No DNA digestion or denaturation—preserve cell morphology, antigens, and dsDNA integrity
  • Streamlined protocol—fewer steps to results
how it works
EdU is a nucleoside analog of thymidine, and is incorporated into DNA during active DNA synthesis. Detection is based on a copper-catalyzed covalent “click” reaction between the alkyne present on EdU and the azide present on the Alexa Fluor® dye. The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, in contrast to BrdU assays that require DNA denaturation (typically using HCl, heat, or digestion with DNase).

HCS CellMask™ Blue Staining

Effect of Click-iT® reaction rinse buffer on HCS CellMask™ blue staining in U2OS cells.
(A) No rinse buffer was used. (B) Click-iT® reaction rinse buffer was used.
Quantity Cat. No.
Click-iT® EdU Alexa Fluor® 647 Imaging Kit *for 50 coverslips* 1 kit C10340
Click-iT® EdU Alexa Fluor® 488 HCS Assay *2-plate size* 1 kit C10350
Click-iT® EdU Alexa Fluor® 488 HCS Assay *10-plate size* 1 kit C10351
what it is
pHrodo™ dextran is a superior alternative to other fluorescent dextran conjugates (e.g., BCECF and tetramethylrhodamine (TRITC)) for live-cell imaging of endocytosis.

what it offers

  • Content-rich results—red-orange fluorescence facilitates multiplexing with blue, green, and far-red fluorescent fluorophores
  • Simple method—minimal fluorescent signal at neutral pH eliminates quenching reagents and extra wash
how it works
pHrodo™ dextran has a pH-sensitive fluorescence emission (excitation and emission maxima of 560/585 nm) that increases in intensity with increasing acidity. This increase is particularly dramatic in the pH range of 4 to 8, commonly seen as endocytic vesicles are acidified. pHrodo™ dextran is essentially dark in the extracellular environment. Upon internalization, the acidic environment of the endosome elicits a bright, red-fluorescent signal that can be visualized by fluorescence microscopy, flow cytometry, or high-content imaging and analysis.

pHrodo Dextran

Multiplexed analysis with pHrodo™ dextran for endocytosis. HeLa cells were transduced with Organelle Lights™ Endosome-GFP. The following day, medium was replaced with serum-free medium plus 50 µM deferoxamine. Cells were washed and incubated with 1 µg/mL Hoechst 33342, 10 µg/mL pHrodo™ Dextran, and Alexa Fluor® 647 transferrin for 5 min at 37°C. (A) Organelle Lights™ Endosome-GFP; (B) Alexa Fluor® 647 transferrin; (C) pHrodo™ dextran.
Quantity Cat. No.
pHrodo™ dextran for endocytosis 0.5 mg P10361
what it is
The CyQUANT® Direct assay is a fluorescence-based cell proliferation and cytotoxicity assay that is well suited for high-throughput screening applications.

what it offers

  • Convenience—no washes, cell lysis, or temperature equilibrations required
  • Accuracy—cytotoxicity data compare well to metabolism-based assays
  • Robust results—readout is independent of metabolic state
how it works
The CyQUANT® Direct assay is based on a cell-permeant DNA-binding dye in combination with a masking dye. The masking dye blocks staining of dead cells and cells with compromised cell membranes so that only viable cells fluoresce. Because DNA content is highly regulated, cell number estimates using the CyQUANT® Direct assay are very accurate.

CyQUANT Direct Assay Protocol

CyQUANT® Direct assay protocol. The CyQUANT® Direct assay is designed for use with multi-well plates (96-, 384-, or 1,536-well formats), making it ideal for high-throughput screening applications. The reagent is added directly to cells in complete medium and incubated for 30 to 60 min. Samples are read from the bottom on standard fluorescence plate readers set for green fluorescence (e.g., Alexa Fluor® 488, fluorescein, fluo-4, and GFP).
Quantity Cat. No.
CyQUANT® Direct Cell Proliferation Assay 1 kit C35011
CyQUANT® Direct Cell Proliferation Assay 1 kit C35012
what it is
Vybrant® DyeCycle™ Ruby Stain is a near-infrared emission stain for DNA content analysis, enabling live cell cycle analysis with limited cytotoxicity.

what it offers

  • Live cell cycle analysis
  • Limited cell toxicity offers cell sorting potential
  • Compatible with any flow cytometer

how it works
The Vybrant® DyeCycle™ Ruby Stain penetrates the cell membrane of intact cells and binds covalently to the DNA in the nucleus. Staining is simple—suspended cells are incubated with Vybrant® DyeCycle™ Ruby Stain and fluorescence is measured directly. No additional treatment or centrifugation is required. This dye takes advantage of the commonly available 488 nm and 633/635 nm excitation sources with emission >670 nm, leaving common blue laser channels open for other studies. And unlike DRAQ5, Vybrant® DyeCycle™ Ruby Stain does not kill the analyzed cells, offering the possibility of cell sorting based on DNA content.

Excitation and emission spectra of Vybrant® DyeCycle™ Ruby Stain.
Quantity Cat. No.
Vybrant® DyeCycle™ Ruby Stain 400 assays
Vybrant® DyeCycle™ Ruby Stain 100 assays
what they are
Invitrogen offers one of the most extensive portfolios of antibodies for cell junctions and the cytoskeleton. We have released new Alexa Fluor® 488 conjugates for key cell junction and cytoskeletal targets.

what they offer

  • Save time and costs by eliminating secondary antibody detection
  • Ideal for imaging multiple targets in colocalization studies
  • Validated against multiple applications
how they work
Alexa Fluor® 488 (excitation/emission 494/517 nm) is one of the brightest dyes available for imaging applications. Its conjugation to highly specific antibodies results in bright, crisp images with almost no background, and bypasses the need for secondary antibody detection. The new Alexa Fluor® conjugates of antibodies against occludin, claudin-1, claudin-4, claudin-5, and α–tubulin are valuable tools for imaging tight junctions and the cytoskeleton, allowing easy colocalization with other key markers.

  • Learn More about the Alexa Fluor® 488 Dye

Human Caco-2 cells stained with mouse anti-α–tubulin Alexa Fluor® 488, showing different phases of mitotic cell division. DNA is counterstained with blue Hoechst 33258.


Product Species reactivity Applications Quantity Cat. No.
Mouse anti-claudin-1,
Alexa Fluor® 488 conjugate
Cn, Hu, Ms, Rt ELISA, ICC, IF, IP, WB 100 µg 374988
Mouse anti-claudin-4,
Alexa Fluor® 488 conjugate
Cn, Hu, Ms, Rt ICC, IF 100 µg 329488
Mouse anti-claudin-5,
Alexa Fluor® 488 conjugate
Hu, Ms, Rt ELISA, ICC, IF, IP, WB 100 µg 352588
Reactivity: Cn, canine; Hu, human; Ms, mouse; Rt, rat
ICC, immunocytochemistry; IF, immunofluorescence; IP, immunoprecipitation; WB, western blot.
what they are
Molecular Probes® Organelle Lights™ reagents are prepackaged and ready-to-use fluorescent protein constructs fused with signal peptides for accurate and specific targeting to subcellular compartments and structures. The collection of Organelle Lights™ reagents has recently expanded to include new colors for staining lysosomes, mitochondria, endoplasmic reticulum (ER), and endosomes.

what they offer

  • Ready-to-use reagents—no potentially harmful cell treatments required
  • Simple method—optimize transduction conditions only once per cell type
  • Versatility—multiplex with other stains in live or fixed cells
how they work
Organelle Lights™ reagents consist of suspensions of baculovirus carrying an expression construct with a fluorescent protein fused to either a cellular protein or a localization peptide. Simply add the virus suspension to your cells, incubate overnight, and you’re ready for imaging. Organelle Lights™ reagents can label a broad range of mammalian cell types, including primary and stem cells, without the need for lipids or dye-loading protocols that can perturb cell growth and viability.

  • Learn More about Organelle Lights™ Reagents
Organelle Lights™ ER-RFP

Live-cell imaging with Organelle Lights™ ER-RFP.  HeLa cells were transduced with Organelle Lights™ ER-RFP. The next day, cells were co-stained with 50 nM MitoTracker® Deep Red 633 and 1 µg/mL Hoechst 33342 (stains nuclei). Imaging was performed on live cells using a DeltaVision® Core microscope and standard DAPI/TRITC/Cy®5 dye filter sets.
Product Quantity Cat. No.
Organelle Lights™ Lysosomes-GFP 1 kit O36228
Organelle Lights™ Mito-RFP 1 kit O36229
Organelle Lights™ ER-RFP 1 kit O36230
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

  • Ready-to-use—no need to purify plasmids or worry about vector integrity
  • Cell-friendly—no harmful treatments are required; primary and stem cells are labeled without apparent cytopathic effects
  • Titratable—defined reagent concentrations enable precise titration and optimization of expression levels
how it works
The Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) is 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.

Premo FUCCI Cell Cycle

Imaging cell cycle progression in live cells with Premo™ FUCCI Cell Cycle Sensor. (A) Schematic of cell cycle progression with nuclear fluorescence changes. (B) U2OS cells were transduced with Premo™ FUCCI Cell Cycle Sensor, then stained with Alexa Fluor® 647 wheat germ agglutinin.

Quantity Cat. No.
Premo™ FUCCI Cell Cycle Sensor 1 kit P36232
what it is

The Countess™ Automated Cell Counter uses trypan blue staining combined with a sophisticated image analysis algorithm to provide accurate cell and viability counts in just 30 seconds. The instrument 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.

what it offers

  • Accurate—eliminate the subjectivity and variability of manual cell counting
  • Fast—count live and dead cells,and measure viability and average cell size in 30 seconds with just 10 μL of sample
  • Convenient—no setup, cleaning, or service required
how it works

The Countess™ Automated Cell Counter can accurately count blood cells including peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs), and white blood cells (WBCs) from lysed whole blood and Ficoll preparations. Because the Countess™ instrument uses trypan blue staining, the viability of WBCs is easily determined, while platelets and noncellular debris are unstained and excluded from the count. The Countess™ instrument does not differentiate between WBC types, and viability information is not available for RBCs.

Countess Cell Counter

The Countess™ Automated Cell Counter

Product Quantity Cat. No.
Countess™ Automated Cell Counter each C10227

Countess™ Automated Cell Counter Starter Kit

1 kit C10310
Countess™ Cell Counting Chamber Slides 50 slides (100 counts) C10228

what it is
LIVE/DEAD® cell viability assays are used to differentially stain live and dead cells in a variety of mammalian cell types. These cells can then be fixed with formaldehyde for subsequent analysis by flow cytometry. The LIVE/DEAD® Fixable Dead Cell Stain Sampler Kit contains all 8 LIVE/DEAD® reactive dyes that are excited with laser lines ranging from UV to far red, making it easier to identify the optimal dead cell stain for multicolor panels.

what it offers

  • Post-fixation analysis—staining pattern preserved
  • Accuracy—eliminates staining artifacts
  • Flexibility—broad dye selection

how it works
LIVE/DEAD® cell viability assays employ the reaction of a fluorescent reactive dye with amines on cellular proteins. Viable cells exhibit faint staining on the cell surface, whereas the dye penetrates the interior of dead cells, resulting in at least a 50-fold increase in fluorescence compared to live cells. Because the dye reacts covalently with proteins, the staining pattern is completely preserved following sample fixation with formaldehyde.

Live-cell gating using LIVE/DEAD® Fixable Violet dye eliminates staining artifacts from analysis. In a comparison between live-cell gating using scatter (A), and live-cell gating using LIVE/DEAD® Fixable Violet dye (B), staining artifacts using scatter gating are illustrated. The significant number of dead cells in subsequent analysis using scatter (C) are noted, as compared to the use of the LIVE/DEAD® Fixable Violet dye (D) to eliminate dead cells. Reproduced with permission from Elsevier ((J Immunol Methods 313:199 (2006)).

Quantity Cat. No.
LIVE/DEAD® Fixable Dead Cell Stain Sampler Kit 1 kit L34960

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