Here's what's in this issue:

Measure cell health and toxicity with high-content imaging and analysis assays

HCS Mitochondrial Health Kit
HCS DNA Damage Kit

what they are
Two new assays for automated imaging platforms provide multiparametric analysis of important biological events related to cell stress, damage, and death.

how they work
The HCS DNA Damage Kit measures DNA damage using antibody-based detection of phosphorylated H2AX (Ser139) in nuclei. The HCS Mitochondrial Health Kit measures mitotoxicity with the MitoHealth stain, which accumulates in the mitochondria of live cells in an amount proportional to the mitochondrial membrane potential. Both kits include the Image-iT® DEAD Green™ Viability Stain for measuring cytotoxicity; drugs and test compounds that cause serious cell injuries, including plasma membrane permeability, allow entry of the dye.

what they offer
  • comprehensive analyses of cell health and toxicity—simultaneous detection of prelethal and lethal aspects of cellular stress and viability
  • content-rich results—both assays include reagents compatible with fixation and detergent-based permeabilization, enabling the addition of other antibody detection steps to the assay
  • user-friendly protocols—simple and robust methods

Detection of genotoxicity and cytotoxicity in valinomycin-treated A549 cells using the HCS DNA Damage Kit. A549 cells were treated with 30 μM or 120 μM valinomycin for 24 hr before the HCS DNA Damage Assay was performed. With increasing concentrations of valinomycin, cells showed genotoxic effects as indicated by detection with a pH2AX antibody labeled with Alexa Fluor® 555 goat anti–mouse IgG (orange fluorescence), and cytotoxic effects as indicated by staining with the Image-iT® DEAD Green™ Viability Stain (green fluorescence). Blue-fluorescent Hoechst 33342 was used as a nuclear segmentation tool, and Alexa Fluor® 647 phalloidin was used to visualize F-actin (pseudocolored magenta). The left image shows untreated cells with intact F-actin cytoskeletons and no evidence of cytotoxicity or genotoxicity. The right image shows cells treated with 120 μM valinomycin, which completely disrupted the actin cytoskeletons, increased levels of DNA damage, and compromised plasma membrane integrity.

Products Quantity Cat. no.
HCS Mitochondrial Health Kit, 2-plate size 1 kit H10295
HCS DNA Damage Kit, 2-plate size 1 kit H10292


A powerful tool for measuring cytotoxicity that survives fixation and permeabilization

Image-iT® DEAD Green™ Viability Stain

what it is
The Image-iT® DEAD Green™ Viability Stain not only measures cytotoxicity, but survives fixation and detergent-based permeabilization, making it ideal for use in HCS assays and standard fluorescence microscopy imaging applications.

how it works
Cytotoxicity is a multifaceted process that often leads to compromised plasma membrane integrity. The Image-iT® DEAD Green™ Viability Stain is impermeant to healthy cells, and can enter cells only after plasma membrane integrity has been compromised. In contrast to viability stains like propidium iodide, the Image-iT® DEAD Green™ Viability Stain is compatible with fixation and permeabilization, enabling multiparametric analyses with other biomarkers or cytotoxicity indicators.

what it offers
  • content-rich results—survives fixation and detergent permabilization, enabling subsequent multiparametric analyses with antibodies
  • simple protocol—add to cells, then incubate

  Cytotoxicity measured using the Image-iT® DEAD Green™ Viability Stain. HeLa cells were treated with DMSO (untreated) or 60 μM valinomycin for 18 hr, stained with Image-iT® DEAD Green™ Viability Stain for 30 min, fixed, and imaged using the Thermo Scientific Cellomics® ArrayScan® VTI Platform.

Product Quantity Cat. no.
Image-iT® DEAD Green™ Viability Stain, 1 mM solution in DMSO 25 μl I10291


Visualize protein expression in cells

Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay

what it is
The Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay provides a fast, sensitive, nontoxic, and nonradioactive method for the detection of nascent protein synthesis using fluorescence microscopy and high-content imaging.

how it works
Click-iT® AHA is an amino acid analog of methionine containing an azido moiety. Similar to 35S-methionine, Click-iT® AHA is added to cultured cells, then incorporated into proteins during active protein synthesis. Detection of the incorporated amino acid uses a chemoselective ligation or “click” reaction between an azide and an alkyne, where the azido-modified protein is detected with Alexa Fluor® 488 alkyne.

what it offers
  • the first imaging assay for detecting newly synthesized proteins or changes in protein expression
  • validated in A549 and U2OS cells with reagents that inhibit protein synthesis, including cycloheximide, anisomycin, and puromycin, in both dose response and min/max format
  • content-rich results—compatible with fixation and detergent-based permeabilization, enabling the addition of other antibody detection steps to the assay

  Dose response curves performed in duplicate. U2OS cells were treated in L-methionine–free media and 50 μM Click-iT® AHA for 30 min with anisomycin (17 pM–1 μM). Cells were washed, fixed, permeabilized, and nascent protein synthesis was detected following a click reaction with Alexa Fluor® 488 alkyne. Imaging and analysis was performed using the Thermo Scientific Cellomics® ArrayScan® VTI Platform.

Product Quantity Cat. no.
Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay, 2-plate size 1 kit C10289

Image late-stage apoptosis before cells “pop”

Click-iT® TUNEL Imaging Assays

what they are
The Click-iT® TUNEL Imaging Assays can detect a higher percentage of apoptotic cells than assays that use other modified nucleotides under identical conditions. The assay is complete within 2 hours and allows multiplexing with surface and intracellular biomarker detection.

how they work
The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay is based on the incorporation of modified dUTPs by the enzyme terminal deoxynuclotidyl transferase (TdT) at the 3’-OH ends of fragmented DNA, a hallmark as well as the ultimate determinate of apoptosis. Often at this final stage of apoptosis, adherent cells are known to detach or “pop” off. For a reliable and reproducible TUNEL imaging assay, it is vital that the modified nucleotide is an acceptable substrate for TdT, and that the detection method is sensitive without causing any additional cell loss. The Click-iT® TUNEL Imaging Assays use dUTP modified with an alkyne, a small, bio-orthogonal functional group that allows the nucleotide to be more readily incorporated by TdT than other modified nucleotides are. Detection is based on a click reaction, a copper (I) catalyzed reaction between an azide and alkyne. Click chemistry fills the void when methods such as direct labeling or the use of antibodies are not efficient. The small size of the Alexa Fluor® azide (MW <~1000) compared to that of an antibody (MW ~150,000) enables easy penetration of complex samples, with only mild fixation and permeabilization required.

what they offer
  • detection of a higher percentage of apoptotic cells under identical assay conditions compared with fluorescein-dUTP or BrdUTP
  • results within 2 hr
  • accurate, consistent performance with content-rich results

  DNA strand breaks typical of late-stage apoptosis visualized using the Click-iT® TUNEL Imaging Assay. HeLa cells were treated with staurosporine, then fixed and permeabilized. The Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay (Cat. no. C10247) was performed. Activated caspase-3 was detected with a polyclonal rabbit primary antibody for cleaved caspase-3 and labeled with Alexa Fluor® 488 goat anti–rabbit IgG (Cat. no. A11008, green fluorescence). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor® 555 goat anti–mouse IgG (Cat. no. A21422, orange fluorescence). Nuclei were stained with Hoechst 33342 (Cat. no. H1399, blue fluorescence). The coverslips were mounted in ProLong® Gold antifade reagent (Cat. no. P36930) before imaging. The cell on the right not only has a high level of caspase-3 activity and DNA strand breaks, but also shows a loss of structural integrity consistent with a cell undergoing apoptosis.

Product Quantity Cat. no.
Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay, for microscopy and HCS 50–100 assays C10245
Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay, for microscopy and HCS 50–100 assays C10246
Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay, for microscopy and HCS 50–100 assays C10247

Primary antibodies against ATP synthase (Complex V) and other mitochondrial proteins

Validated primary antibodies for mitochondrial research

what they are
Invitrogen offers the most complete portfolio of antibodies for mitochondrial research, including antibodies against ATP synthase (Complex V). New this month are antibodies against nicotinamide nucleotide transhydrogenase (NNT) and mitochondrial trifunctional protein (TFP), as well as the ApoTrack™ Cytochrome c WB Cocktail for detecting apoptosis.

how they work
The most important function of mitochondria is production of ATP; deficiencies in this process result in many genetic and neurological diseases. Complex V, also called F 1F 0-ATPase and ATP synthase, is responsible for ATP production in oxidative phosphorylation, and can work in reverse as a proton-pumping ATPase. The enzyme in mammals is composed of 17 subunits, 5 of which make up the easily detached F 1. The remainder are components of two-stalk domains and the proton-pumping F 0 part of the machinery. Invitrogen offers antibodies against subunits of ATP synthase as well as other mitochondrial proteins that work closely with the electron transport chain—including new antibodies against nicotinamide nucleotide transhydrogenase (NNT) and mitochondrial trifunctional protein (TFP). Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial inner membrane protein catalyzing the interconversion of NADH and NADPH within the matrix space of the organelle. NNT couples this reaction to proton translocation across the inner membrane. Mitochondrial trifunctional protein (TFP), located in the inner mitochondrial membrane, is part of the mitochondrial fatty acid beta-oxidation pathway. TFP catalyzes three out of the four steps in the beta-oxidation cycle, with a specific affinity for long chain substrates. Inherited deficiency of TFP is a recessive genetic disease that occurs in approximately 1:38,000 newborns in the US. Deficiency of LCHAD (the dehydrogenase part of TFP) has also been found in children of women who develop HELLP syndrome (1:1,000 pregnancies) and AFLP syndrome (1:13,000 pregnancies), both of which are life-threatening obstetric conditions. For mitochondrial researchers studying apoptosis, the ApoTrack™ Cytochrome c WB Cocktail provides a method for detecting the release of cytochrome c and other apoptogenic proteins from the mitochondrial intermembrane space into the cytoplasm.

what they offer
  • the best specificity for mitochondrial proteins
  • validated reagents with multiple applications and species specificities
  • part of an extensive portfolio of antibodies for mitochondrial research

  Staining of HDFn cells. HDFn cells were detected with nicotinamide nucleotide transhydrogenase (NNT) monoclonal antibody (Cat. no. 459170) and labeled with Alexa Fluor® 594 goat anti–mouse IgG1 secondary antibody (Cat. no. A11005).

Product Reactivity Application Quantity Cat. no.
ApoTrack™ Cytochrome c WB Cocktail Hu WB 100 μg 459160
Nicotinamide Nucleotide Transhydrogenase (NNT) monoclonal antibody (WB and ICC) Hu, B, Rt, Ms WB, ICC 100 μg 459170
Mitochondrial Trifunctional Protein (MTP) monclonal antibody (WB) Hu WB, IP 180 μg 459180
Complex V d-subunit Monoclonal Antibody Hu, Ms, B, Rt, Ce WB, IF 100 μg 459000
Complex V immunocapture monoclonal antibody Hu, B, Rt, Ms IP 100 μg 439700
Complex V subunit alpha monoclonal antibody Hu, B WB, ICC 100 μg 439800
Complex V subunit F6 monoclonal antibody Hu, B, Ms WB, ICC 100 μg 439900
Complex V Mitoprofile® Immunocapture Kit Ms, Rt IP 100 μg 457425
Hu = human; B = bat; Rt = rat; Ms = mouse; Ce = C. elegans.

For a complete list of mitochondrial research antibodies, visit www.invitrogen.com/antibodies.

A robust and sensitive chloride assay

Premo™ Halide Sensor

what it is
The Premo™ Halide Sensor was developed for functional studies of ligand- and voltage-gated chloride channels. Transient BacMam-mediated expression of a halide-sensitive yellow fluorescent protein (YFP) mutant is efficient in a range of cell types, including human primary cells.

how it works
The combination of BacMam delivery, the bright fluorescence of the Venus variant of Aequorea victoria green fluorescent protein (GFP), and a quenching iodide surrogate ion provides a highly sensitive assay for robust and convenient screening of halide ion channels and transporter modulators in your preferred mammalian cellular model.

what it offers

  • chloride channel–specific—measure halide ion flux in voltage- and ligand-gated chloride channels and transporters
  • robust—reliably high expression, no-wash format, excellent signal window, and compatibility with most fluorescent HTS readers
  • flexible—study chloride flux in your cellular model with efficient and noncytopathic delivery of Premo™ Halide Sensor by BacMam technology
  • pharmacologically relevant—known modulators show dose-dependent quenching, and BacMam delivery enables assays in primary cells


Principle of the Premo™ Halide Sensor. Iodide redistribution upon chloride channel activation. Basal fluorescence from Premo™ Halide Sensor is high when chloride channels are low. Upon activation (opening) of chloride channels, the iodide ions enter the cell down its concentration gradient, quenching the fluorescence from the Premo™ Halide Sensor.

Product Quantity Cat. no.
Premo™ Halide Sensor for 10 microplates 1 kit P10229


Learn more about the Premo™ Halide Sensor

Directly labeled primary antibodies for flow cytometry

Fluorescent dye–labeled primary antibodies

what they are
Invitrogen offers new primary antibodies conjugated to Qdot® nanocrystals, Alexa Fluor® dyes, Pacific Blue™ dye, and Pacific Orange™ dye, with validation for flow cytometry.

how they work
Directly conjugated primary antibodies are available in a variety of fluorescent colors to maximize multicolor flow cytometric analysis. These antibodies give researchers the tools to find answers to complex biological questions relating to cancer, immunology, stem cell research, and activation studies. Simultaneous measurement of different specificities reveals more information about individual cells in less time, with less sample.

what they offer
  • more options for multicolor analysis
  • greater flexibility and precision in designing multicolor experiments
  • tools to satisfy the growing need for more information per sample

To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.
To find antibodies and other reagents for flow cytometry, visit www.invitrogen.com/flowcytometry.

  Ntera2 cells stained with Alexa Fluor® 647 dye–conjugated anti-SSEA4 monoclonal antibody (Cat. no. SSEA421).  The negative profile represents unstained cells.


Product Regulatory status Size Cat. no.
CD1a, Mouse anti-human Alexa Fluor® 488 ASR 0.5 ml CD1a20
CD2, Mouse anti-human, Qdot® 605 conjugate* RUO 100 μl Q10172
CD3, Hamster anti-mouse PE-Alexa Fluor® 700 RUO 1.0 ml CD0324
CD8, Mouse anti-human, Qdot® 565 conjugate* RUO 100 μl Q10152
CD10, Mouse anti-human, Qdot® 605 conjugate* RUO 100 μl Q10153
CD15, Mouse anti human Pacific Blue™ ASR 0.5 ml MHCD1528
CD16, Mouse anti-human Pacific Blue™ ASR 0.5 ml MHCD1628
CD16, Mouse anti-human Pacific Orange™ ASR 0.5 ml MHCD1630
CD19, Mouse anti-human, Qdot® 655 conjugate* RUO 100 μl Q10179

CD20, Mouse anti-human Pacific Blue™

ASR 0.5 ml MHCD2028
CD23, Mouse anti-human Alexa Fluor® 488 ASR 0.5 ml MHCD2320
CD29, Mouse anti-human Alexa Fluor® 488 ASR 0.5 ml CD2920
CD45, Rat anti-mouse Pacific Blue™ RUO 1.0 ml/100 μg MCD4528
CD45R(B220), Rat anti-mouse, Qdot® 655 conjugate* RUO 100 μl Q10176
CD56, Mouse anti-human Alexa Fluor® 488 ASR 0.5 ml MHCD5620
CD69, Hamster anti-mouse Pacific Blue™ RUO 1.0 ml/100 μg HM4028
CD105, Mouse anti-human Alexa Fluor® 488 ASR 0.5 ml MHCD10520
HLA-DR, Mouse anti-human Pacific Blue™ ASR 0.5 ml MHLDR28
HLA-DR, Mouse anti-human, Qdot® 605 conjugate* RUO 100 μl Q10052
HLA-DR, Mouse anti-human, Qdot® 800 conjugate* RUO 100 μl Q10063
IFN-Gamma, Mouse anti-human Pacific Blue™ RUO 0.5 ml MHCIFG28
Mouse anti-SSEA4 Alexa Fluor® 647 RUO 0.5 ml SSEA421
Mouse anti-SSEA4 Alexa Fluor® 700 RUO 0.5 ml SSEA429
Hamster IgG Isotype control PerCP RUO 0.5 ml/ 50 ug HM31
Hamster IgG Isotype control PE-Alexa Fluor® 700 RUO 0.5 ml HM24
* Qdot conjugates are optimally excited by UV lasers or 405 nm light, but may be excited by other lasers as well.

proven performers

Zenon® labeling technology

what it is
Zenon® labeling technology provides a rapid, versatile, and reliable method for producing fluorescently labeled antibodies using a variety of fluorochromes, even with small amounts of unpurified starting material such as hybridoma culture supernatant. Antibodies labeled using Zenon® labeling technology are suitable for use in many applications where a directly labeled antibody can be used. Zenon® labeling technology provides a versatile, easy-to-use system for labeling human, mouse IgG1, IgG2a, and IgG2b antibodies, and rabbit IgG antibodies.

how it works
Zenon® fragments are specifically designed to target and bind to the Fc portion of the primary antibody only, giving a rapid, noncovalent method of quickly labeling small quantities of primary antibody. Zenon® labeling technology is simple and efficient; the entire labeling procedure takes only 10 min. Once the Zenon® antibody complex has formed, it can be stored until needed; just before use, nonspecific IgG is added to block any unbound Zenon® fragments, making column purification unnecessary. The Zenon® complex can then be applied directly to the sample.

what it offers
  • 10-min labeling time
  • 1–20 µg primary antibody per labeling
  • compatible with BSA and other stabilizing proteins
  • no purification step
  • flexible antibody labeling

Now through March 31, 2009, visit www.invitrogen.com/zenon and take a short survey to receive 25% off your next purchase of Zenon® products.


How Zenon® labeling technology works. Noncovalent labeling with Zenon® antibody labeling vs. covalent labeling with the Microscale, Monoclonal, and Protein Labeling Kits.

buzzworthy

Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-κB–regulated antiapoptotic and cell survival gene products in human multiple myeloma cells.
Bhardwaj, A. et al. (2007) Blood 109: 2293–2302.

Can natural products help outwit cancer cell defenses? The potential anticancer benefits of resveratrol—a naturally occurring polyphenol abundant in red grapes—have been demonstrated in a wide variety of tumor cells. However, its effect in multiple myeloma (MM) cells, which can be particularly resistant to existing chemotherapies, has not been investigated. Chemoresistance in MM cells has been shown to involve numerous mechanisms, including overexpression of antiapoptotic substances and activation of cell survival pathways. In the present study, the authors examined the effect of resveratrol treatment on the proliferation and antiapoptotic potential of drug-sensitive and drug-resistant MM cells. They observed significant suppression of MM cell proliferation in response to resveratrol treatment, as well as potentiation of the apoptotic effect of two drugs widely used to treat MM, as revealed through use of the Molecular Probes® LIVE/DEAD® assay. Resveratrol treatment also caused MM cells to accumulate in the sub-G1 cell-cycle phase, accompanied by an increase in apoptosis-related substances and measurable downregulation of antiapoptotic gene products. Significantly, their results showed that resveratrol inhibited both inducible and constitutive NF-κB activity, and suppressed several genes whose products are known to contribute to oncogenesis. Their results offer the first evidence that resveratrol is effective in overcoming chemotherapy-induced resistance in MM cells, offering promise for its potential use to enhance the efficacy of standard chemotherapeutic agents.

View bibliography reference

the view

LCRMP-1 is a novel isoform of collapsin response mediator protein-1 (CRMP-1) that promotes cancer cell invasion and metastasis. To investigate whether the effect of LCRMP-1 on cancer cell invasion and metastasis is related to actin filaments, colocalization of LCRMP-1 and actin filaments was examined. Plasmid pEGFP-LCRMP-1 was transfected into human lung adenocarcinoma CL1-5 cells using 20 U of Lipofectamine™ reagents (Life Technologies) in 1 ml of Opti-MEM® medium (Life Technologies). After 24 hr, live cells were washed with PBS, incubated in 3.7% cold paraformaldehyde for 10 min, and permeabilized in a Triton® solution for 10 min at room temperature. Samples were blocked with 3% BSA and stained with 5 U/ml of Alexa Fluor® 594 phalloidin (Life Technologies) for 30 min. Following additional washes with PBS, the coverslips were mounted on slides using antifade mounting solution (Life Technologies) and visualized using a Zeiss Axiophot epifluorescence microscope equipped with an MRC-1000 laser-scanning confocal imaging system (Bio-Rad Laboratories). The image shows exogenous GFP-LCRMP-1 fusion proteins colocalized with stained F-actin in CL1-5 cells, especially in the lamellipodia and filopodia. This also indicates that cells that exhibit highly invasive phenotypes may have more filopodia and lamellipodia. Image submitted by Si-Hua Pan, National Defense Medical Center, Taiwan.


on the web

  Find products for the integrin pathway
Empower your research today with Invitrogen’s comprehensive portfolio of products and services for investigating integrin signaling. You'll find everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, as well as world-class profiling and screening services. View our portfolio of reagents for integrin signaling at www.invitrogen.com/integrin.

 

in the field

Calling all HCA researchers
Join Invitrogen at the upcoming High-Content Analysis meeting hosted by the Cambridge Healthtech Institute at The Fairmont Hotel in San Francisco, California, USA, January 6–9, 2009. Along with leaders from across the industry, we will be presenting our latest tools and assays for high-content analysis platforms. Stop by and see us at booth #14.



Molecular Probes® The Handbook

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cellular imaging │high-content screening │ HCS Mitotic Index Kit

Cell health and toxicity in high-content imaging and analysis

what it is
The HCS Mitotic Index Kit is a powerful image-based assay for the identification of compounds that affect mitotic progression.  

how it works
The HCS Mitotic Index Kit includes a primary antibody against phosphorylated histone H3 (Ser10) as a sensitive index of mitosis, and a secondary antibody conjugated to the green-fluorescent Alexa Fluor® 488 dye. Blue-fluorescent DAPI and near-infrared–fluorescent HCS NuclearMask™ Deep Red stain, both included in the kit, provide two choices for DNA profiling and cell demarcation for image analysis.

what it offers
  • comprehensive analyses of cell health and toxicity—simultaneous detection of prelethal and lethal aspects of cellular stress and viability
  • content-rich results—both assays include reagents compatible with fixation and detergent-based permeabilization, enabling the addition of other antibody-based parameters to the assay
  • simple, user-friendly protocol

HCS Mitotic Index  

Dose response for nocodazole in A549 cells using the HCS Mitotic Index Kit. A549 cells were treated with nocodazole at final concentrations between 0 to 1,000 nM and incubated for 24 hr. Imaging and analysis were performed using a 10X objective and the Compartmental Analysis BioApplication with the Thermo Scientific Cellomics® ArrayScan® VTI platform.


Product Quantity Cat. no.
HCS Mitotic Index Kit 1 kit H10293