ProbesOnline, July 2009

In This Issue

FEATURED NEW PRODUCTS

what it is
Chemiluminescent Alkaline Phosphatase ELISA Kits are used for rapid, ultrasensitive antigen detection in enzyme immunoassays employing an alkaline phosphatase label. The kits are available with a choice of ready-to-use substrate/enhancer formulations, a blocking reagent, and buffer. A sampler kit containing all four substrate/enhancer formulations is available to facilitate assay development. The use of chemiluminescence significantly extends the limit of detection compared to colorimetric, fluorometric, and isotopic methods.



what it offers

  • Wide dynamic range
  • High sensitivity
  • Compatibility with multiple assay formats


how it works
This detection system incorporates CSPD® or CDP-Star® 1,2-dioxetane alkaline phosphatase substrates with the luminescence enhancers Emerald-II™ or Sapphire-II™ for analyte detection in enzyme-linked immunoassays. 1,2-dioxetane substrates emit visible light upon enzyme-catalyzed decomposition, which is quantitated with a simple luminometer. The addition of macromolecular luminescence enhancers increases the emission efficiency of light production by partitioning water away from the site of signal production.

Comparison of Chemiluminescent, Fluorescent & Colorimetric

Comparison of chemiluminescent, fluorescent, and colorimetric detection for ELISA quantitation of rhIL-6.
Product Quantity Cat. No.  
Chemiluminescent Alkaline Phosphatase ELISA Kit #1: with CSPD® substrate and Sapphire-II™ Enhancer1,000 assaysC10552Order Now
Chemiluminescent Alkaline Phosphatase ELISA Kit #2: with CSPD® substrate and Emerald-II™ Enhancer1,000 assaysC10553Order Now
Chemiluminescent Alkaline Phosphatase ELISA Kit #3: with CDP-Star® substrate and Sapphire-II™ Enhancer1,000 assaysC10554Order Now
Chemiluminescent Alkaline Phosphatase ELISA Kit #4: with CDP-Star® substrate and Emerald-II™ Enhancer1,000 assaysC10555Order Now
Chemiluminescent Alkaline Phosphatase ELISA Sampler Kit1,000 assaysC10556Order Now

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.
Product Quantity Cat. No.  
Click-iT® EdU Alexa Fluor® 488 Imaging Kit *for 50 coverslips*1 kitC10337Order Now
Click-iT® EdU Alexa Fluor® 594 Imaging Kit *for 50 coverslips*1 kitC10339Order Now
Click-iT® EdU Alexa Fluor® 647 Imaging Kit *for 50 coverslips*1 kitC10340Order Now
Click-iT® EdU Alexa Fluor® 488 HCS Assay *2-plate size*1 kitC10350Order Now
Click-iT® EdU Alexa Fluor® 488 HCS Assay *10-plate size*1 kitC10351Order Now
Click-iT® EdU Alexa Fluor® 594 HCS Assay *2-plate size*1 kitC10354Order Now
Click-iT® EdU Alexa Fluor® 594 HCS Assay *10-plate size*1 kitC10355Order Now
Click-iT® EdU Alexa Fluor® 647 HCS Assay *2-plate size*1 kitC10356Order Now
Click-iT® EdU Alexa Fluor® 647 HCS Assay *10-plate size*1 kitC10357Order Now

what it is
Click-iT® RNA Alexa Fluor® Imaging Kits and HCS Assays provide the tools necessary to produce quantitative and qualitative measurements of RNA synthesis. The ability to detect newly synthesized RNA or changes in RNA levels, particularly viral RNA, will aid in understanding viral gene expression and genome replication, as well as its subcellular localization in host cells. The small size of the Click-iT® detection molecule allows easy penetration of complex samples. The assay is multiplex-compatible with other probes including antibodies, for the simultaneous detection of RNA-interacting proteins.



what it offers

  • Rapid results—Nascent RNA detection is complete within 30 minutes
  • Content-rich results—multiplex-compatible with other probes
  • Specificity— Efficient incorporation by RNA polymerases but not DNA polymerases

how it works
Utilizing an alkyne-modified nucleoside, 5-ethynyl uridine (EU), and powerful click chemistry, newly synthesized RNA can be detected with a simple, two-step procedure. In step one, the alkyne-containing nucleoside is fed to cells or animals, and is actively and selectively incorporated into nascent RNA. Detection utilizes the chemoselective ligation or “click” reaction between an azide and an alkyne, where the modified RNA is detected with a corresponding azide-containing dye.

RNA Transcription

Subcellular localization of viral RNA transcription in vero cells. Vero cells were pretreated with 2 µM actinomycin D to inhibit host cell transcription and then infected with Tacaribe virus. Infected cells were incubated with 2 mM EU for 1 hr followed by cold methanol fixation and Triton® X-100 permeabilization. Nascent RNA (green) was detected with the Click-iT® RNA Alexa Fluor® 488 imaging kit. Viral nucleoprotein was detected with a directly labeled Alexa Fluor® 594 monoclonal antibody (red). Colocalization of EU and virus nucleoprotein indicated transcription sites in the host cells (yellow).
Product Quantity Cat. No.  
Click-iT® RNA Alexa Fluor® 488 Imaging Kit *for 25 coverslips*1 kitC10329Order Now
Click-iT® RNA Alexa Fluor® 594 Imaging Kit *for 25 coverslips*1 kitC10330Order Now
Click-iT® RNA Alexa Fluor® 488 HCS Assay *2-plate size*1 kitC10327Order Now
Click-iT® RNA Alexa Fluor® 594 HCS Assay *2-plate size*1 kitC10328Order Now

NEW APPLICATIONS

The ability to measure cytoskeletal disruption is an important aspect of cytotoxicity screening in drug discovery and development. While fluorescent conjugates of phalloidin and anti-tubulin antibodies are popular probes used for measuring cytoskeletal disruption, a mask of the entire cell is also important for both cellular demarcation and to determine cell size. Cellular demarcation enables automated image analysis software to determine where the cytoskeleton of one cell ends and another begins, and cannot be done accurately with a nuclear counterstain alone.   

Invitrogen’s HCS CellMask™ stains can be used to measure quantitative differences in cell size resulting from drug treatment. The stains range in fluorescence emission from blue to deep red, allowing researchers flexibility when performing multi-parametric assays for high-content imaging. 

 

Cytochalasin D treatment on HeLa Cells
The effects of cytochalasin D treatment on HeLa cell size as measured by HCS CellMask™ Blue stain. HeLa cells were treated with DMSO vehicle (left) or 10 µM cytochalasin D (right) for 3 hr before fixation and permeabilization. Samples were then labeled with HCS CellMask™ Blue stain, Alexa Fluor® 488–conjugated phalloidin to visualize filamentous actin (red), mouse anti-α-tubulin IgG detected with Alexa Fluor® 555 goat anti-mouse IgG (green), and TO-PRO®-3 iodide to counterstain nuclei (magenta). The bar graph represents quantitative measurements of cell size as indicated by HCS CellMask™ Blue stain to show the effects of cytochalasin D treatment.

 

Product Quantity Cat. No.  
HCS CellMask™ Red stain 1 setH32712Order Now
HCS CellMask™ Orange stain 1 setH32713Order Now
HCS CellMask™ Green stain 1 setH32714Order Now
HCS CellMask™ Blue stain1 setH32720Order Now
HCS CellMask™ Deep Red stain1 setH32721Order Now

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.

Despite being one of the most popular viability stains, trypan blue has recently been recognized as a potentially hazardous chemical. As a result, many labs minimize exposure by working with smaller volumes or lower concentrations of trypan blue. The Countess™ Automated Cell Counter requires only 5 µL of trypan blue and is shipped precalibrated for use with 0.4% trypan blue. By simply and quickly recalibrating the instrument, lower than conventional concentrations of trypan blue (0.1%) can be used for cell counting.

 

Countess Cell Counter 

The Countess™ Automated Cell Counter

 

Product Quantity Cat. No.  
Countess™ Automated Cell CountereachC10227Order Now

Countess™ Automated Cell Counter Starter Kit
(includes 1 cell counter and 11 boxes of slides)

1 kitC10310Order Now
Countess™ Automated Cell Counter Lab Starter Kit
(includes 1 cell counter and 101 boxes of slides)
1 kitC10311Order Now
Countess™ Cell Counting Chamber Slides50 slides (100 counts)C10228Order Now
Countess™ Cell Counting Chamber Slides500 slides (1,000 counts)C10312Order Now
Countess™ Cell Counting Chamber Slides1,250 slides (2,500 counts)C10313Order Now
Countess™ Cell Counting Chamber Slides2,500 slides (5,000 counts)C10314Order Now
Countess™ Cell Counting Chamber Slides5,000 slides (10,000 counts)C10315Order Now

DEPARTMENTS

Buzzworthy

A pHrodo™ Dye–based Method for Monitoring the Phagocytic Internalization of Apoptotic Cells
 
A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.
Miksa M, Komura H, Wu R et al. (2009) J Immunol Methods 342:71–77.

How can we accurately assay the phagocytosis of apoptotic cells?
A key component of apoptosis is the phagocytic removal of cellular debris that results when cells undergo apoptotic self-termination; this clean-up step is carried out by specialized immune cells such as macrophages and neutrophils. While many methods have been developed to assay the initial binding step of phagocytosis, these methods can lead to overestimation of the extent of internalization. In this report, Miksa and colleagues describe a simple and elegant method for monitoring the internalization step itself, which relies on macrophage-mediated chemical alteration of the internal environment of engulfed particles.

Methods
To monitor phagocytosis, target cells (apoptotic thymocytes) were loaded with the pH-sensitive fluorescent dye pHrodo™ Succinimidyl Ester (SE). Upon phagocytic engulfment by macrophages, these target cells are lysed and their contents encounter the acidic environment within the phagosome, causing a dramatic increase in pHrodo™ dye fluorescence. This assay was used to monitor the engulfment of apoptotic thymocytes by FACS flow cytometric analysis in addition to direct observation by fluorescence microscopy.

Results
In addition to showing excellent agreement with previously reported results, the authors accurately characterized the limited phagocytic activity of cells deficient in MFGE8 (a key protein required for phagocytosis), and demonstrated the successful recovery of phagocytic activity upon addition of exogenous MFGE8.

This study represents the first application of this approach to eukaryotic apoptotic cells. The authors suggest that the pHrodo™ dye–based assay may prove useful in future studies of the role of macrophages in apoptosis.

View bibliography reference

 

Product Quantity Cat. No.  
pHrodo™ Succinimidyl Ester (pHrodo™, SE)1 mgP36600pHrodo, SE
Free Online Technical Webinars 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.
Webinars Date Time
Tools for High-Content Screening and Automated Imaging July 21, 2009 10:00 a.m. PT
Fluorescence Immunolabeling Tips and Tricks August 4, 2009 10:00 a.m. PT

Missed our previous webinars? Find our recorded webinars here!




 

Watch the Video of Cellular Lights Histone 2B-RFP Imaging cell division in real time. U2OS cells were transduced with Cellular Lights™ Histone 2B-RFP (red) and Cellular Lights™ MAP4-GFP (green) Cells were kept in a climate-controlled chamber and imaged in McCoy's media with a 40x objective and FITC/TRITC filters. Images were collected every 5 min for 7 hr and 35 min. Time lapse imaging was performed on the DeltaVision® Core microscope.

Watch the video!

Product Quantity Cat. No.  
Cellular Lights™ Histone 2B-RFP1 kitC10129Order Now
Cellular Lights™ MAP4-GFP1 kitC10105Order Now
McCoy’s 5A Medium (modified) (1X), liquid500 mL12330031Order Now


Tracking Tumor Metastasis — DQ™ Collagen

Tumor cells release matrix metalloproteinases (MMPs) including collagenases to facilitate the breakdown of various extracellular matrix (ECM) components. This breakdown allows tumor cells to migrate across cell and tissue boundaries—a process known as metastasis. The fluorescein conjugate DQ™ collagen can be used to directly monitor the collagenase activity of tumor cells. This collagen substrate readily self-quenches, but fluoresces upon cleavage. In combination with CellTracker™ dyes, DQ™ collagen can be used for live-cell tracking of cell migration [1], or for in situ zymography on unfixed cryosections. DQ™ Collagen assays are also amenable to large-scale studies of metastasis inhibitors in drug discovery settings.

1. Sameni et al. (2009) Clin Exp Metastasis 26:299–309.

 

Interaction of Tumor Spheroids
 
Interaction of tumor spheroids with stromal fibroblasts results in extensive degradation of type IV collagen.
HCT116 human colon carcinoma cells were cocultured with human colon fibroblasts prestained with CellTracker™ Orange in reconstituted basement membrane containing DQ™ collagen, type IV. Extensive proteolysis, resulting in fluorescence of DQ™ collagen IV (green), occurs at the site of interaction between the tumor spheroids and the fibroblasts (orange), with yellow regions denoting areas of overlap. Magnification: 40x. Image courtesy of Mansoureh Sameni and Bonnie Sloane, Wayne State University.

 

Product Quantity Cat. No.  
DQ™ collagen, type IV from human placenta, fluorescein conjugate1 mgD12052Order Now
DQ™ collagen, type I from bovine skin, fluorescein conjugate1 mgD12060Order Now
Geltrex™ Reduced Growth Factor Basement Membrane Matrix1 mL12760013Order Now
CellTracker™ Blue CMAC5 mgC2110Order Now
CellTracker™ Green CMFDA20 x 50 μgC7025Order Now
CellTracker™ Orange CMTMR1 mgC2927Order Now
CellTracker™ Orange CMRA20 x 50 μgC34551Order Now
CellTracker™ Red CMTPX20 x 50 μgC34552Order Now

 

Chemiluminescence Assays New Web Resource for Chemiluminescence Assays
Invitrogen offers a wide spectrum of ultrasensitive assays with a chemiluminescent readout. These assays consistently provide high-intensity signal, low background, high sensitivity, wide dynamic range, and rapid results, and are compatible with multiple assay formats (ELISA, reporter gene, and cAMP) under physiologically relevant conditions. Now it’s easier than ever to find the right chemiluminescence assay for your research.

 

 

 Cell and Tissue Analysis Technical Support

  • Have questions about our immunoassay or cellular imaging products?
  • Need help choosing the right assay?
  • Trying to reduce background, or improve sensitivity?

Choose a product area on the Cell and Tissue Analysis Technical Support page to access FAQs, protocols, and troubleshooting assistance.

Browse Online Cell & Tissue Analysis Technical Support

Invitrogen and Zymera Broaden the Use of Qdot® Nanocrystals to Enhance In Vivo Imaging and Biomarker Discovery

Invitrogen has licensed its extensive intellectual property estate related to Qdot® nanocrystal technology to Zymera, a nanobiotechnology company focused on the development of illumination technology for life science research. Zymera will use Qdot® Nanocrystals to create new, self-illuminating quantum dot products to improve in vivo imaging and biomarker discovery, and for the development of new biosensing applications.

Zymera’s novel self-illumination technology uses Bioluminescence Resonance Energy Transfer (BRET) to transfer light from a bioluminescent protein—such as luciferase—directly to quantum dots. First described by the Rao lab at Stanford,1 the resulting BRET dots produce light without an external source of illumination, eliminating autofluorescence background and the need for external light sources, such as lasers. As a result, it is possible to visualize targets deeper in tissue sections or in living animals, and to identify multiple targets simultaneously with a wider variety of detection devices. Zymera expects to combine the technologies to develop new products for tracing blood and lymphatic fluid flow, tracking cells, and detecting biomarkers for use across a range of life science applications.

1. Curr Opin Biotechnol 20:37(2009).



Design of the BRET Qdot Conjugate

Design of the BRET
Qdot® conjugate and the BRET Qdot® catalytic reaction. Exposure to the luciferase substrate, coelenterazine, causes the emission of light of peak wavelength 480 nm from the luciferase enzyme. The energy from this reaction couples non-radioactively to the Qdot® acceptor (shown by the green dotted arrows). In this example, the Qdot® nanocrystal emits light in the red to near-infrared regions (655 nm). The Luc8 enzyme has 8 mutations that confer a 200-fold increase of stability in serum and a 4-fold improvement in light output over the native enzyme.



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