Click-iT® toolbox reagents for rapid detection and identification of posttranslational modifications
 

what they are 
With the latest additions to the Click-iT® toolbox of azide- and alkyne-containing biomolecules, you can rapidly discover key, but often elusive, protein posttranslational modifications (PTMs) including:

  • fucosylation—an important PTM for glycans that participate in cell–cell interactions and cell migration processes
  • prenylation (farnesyl or geranylgeranyl)—important PTMs for protein trafficking
  • fatty acylation (myristic acid or palmitic acid)—important PTMs for cell signaling, protein localization, trafficking, stability, and protein–protein interactions


how they work
Click chemistry is a powerful method for joining biomolecules quickly and specifically, and offers significant advantages over standard metabolic labeling and detection methods. An extremely fast reaction, click chemistry involves a copper-catalyzed covalent reaction between an alkyne and an azide, functional groups that do not occur naturally in biological systems. This uniqueness virtually eliminates the nonspecific binding or high background that can compromise other widely used detection methods. The small size of alkynes and azides compared to antibodies, or even biotin, allows for effective modification of small molecules and efficient penetration of cells and tissue by the Click-iT® reagents. Performing a click reaction requires the corresponding dye- or hapten-azide or alkyne and either the Click-iT® Cell Reaction Buffer Kit for analysis by flow cytometry or imaging, or the Click-iT® Protein Reaction Buffer Kit for analysis by standard biochemistry techniques such as gel electrophoresis, western blotting, or mass spectrometry. 

what they offer

  • pioneering biological insights—expose previously undiscovered and elusive posttranslational modifications
  • rapid results—discover and identify modified proteins in hours or days as opposed to weeks or months with radiolabeling techniques
  • context-rich data—easy multiplexing with other detection methods, including immunolabeling, flow cytometry, and even western blotting

 

Overview of the Click azide/alkyne reaction

Overview of the click chemistry azide/alkyne reaction. The azide and alkyne moieties are interchangeable, where the molecule can be labeled with an alkyne and react with a fluorophore- or hapten-azide.

Product Quantity Cat. no.
Click-iT® fucose alkyne (tetraacetylfucose alkyne)5 mgC10264
Click-iT® geranylgeranyl alcohol, azide *mixed isomers*1 mgC10249
Click-iT® farnesyl alcohol, azide *mixed isomers*1 mgC10248
Click-iT® myristic acid (12-azidododecanoic acid)1 mgC10265
Click-iT® palmitic acid (15-azidopentadecanoic acid)1 mgC10268
Click-iT® Cell Reaction Buffer Kit1 kitC10269
Click-iT® Protein Reaction Buffer Kit1 kitC10276

 

Fluo-4 Direct™ calcium indicator in an “addition-only” format

what it is
The Fluo-4 Direct™ Calcium Assay Kit was formulated to achieve the largest assay window by suppressing background fluorescence generated from media, with little to no impact on the cellular fluorescence. This advanced formulation allows the assay to be run in a simple “addition-only” format, without media removal or wash steps, in the presence of serum-containing media.

how it works
The Fluo-4 Direct™ kit is formulated with a proprietary background fluorescence quencher that eliminates the background fluorescence due to extracellular fluorescent dye and components of the culture media. This eliminates the requirement for media removal and wash steps, and as a result saves time and reduces variability caused by incomplete liquid removal or physical disruption of the cellular monolayer. The Fluo-4 Direct™ assay can be used to obtain appropriate pharmacological profiles for compounds in both agonist and antagonist modes, and is ideal for high-throughput screening experiments.

what it offers
  • compatible with serum-containing media—no removal or wash steps needed
  • simple add-and-read format—just load cells, stimulate, and read
  • large assay window—low background with high fluorescence intensity

Fluo-4 Direct™ Calcium Assay Kit.
Elimination of background fluorescence with the Fluo-4 Direct™ Calcium Assay Kit. M1 CHO cells were plated in media containing 10% FBS and allowed to attach. Cells were loaded with (A) Fluo-4 NW (without media removal) and (B) Fluo-4 Direct™ Calcium Indicator. The background-suppressing capability of the Fluo-4 Direct™ kit leads to elimination of extracellular fluorescence, allowing the cells loaded with Fluo-4 Direct™ indicator to be clearly seen over any residual background fluorescence.

Muscarnic 1 (M1) receptor agonists
Dose-dependent calcium response to muscarnic 1 (M1) receptor agonists.
 Muscarnic 1 (M1) receptor antagonists
Dose-dependent inhibition of the calcium response by M1 receptor antagonists.

Product Quantity Cat. no.
Fluo-4 Direct™ Calcium Assay Kit10 x 10 mL (10 single plates)F10471
Fluo-4 Direct™ Calcium Assay Kit100 mL (10 plates)F10472
Fluo-4 Direct™ Calcium Assay Kit1 L (100 plates)  F10473

To learn more about the Fluo-4 Direct™ Calcium Assay Kit, visit www.invitrogen.com/fluo4direct
or email us at fluo4direct@invitrogen.com.
New dyes for fixed-cell DNA content analysis

what they are
The new FxCycle™ dyes are DNA stains for fixed-cell DNA content analysis, and are alternatives to the 488 nm excitation cell cycle dyes propidium iodide and 7-aminoactinomycin D (7-AAD).  FxCycle™ dyes use either the 405 nm violet or 633 nm red lasers for excitation.

how they work
Measurement of DNA content enables the study of cell populations in various phases of the cell cycle as well as analysis of DNA ploidy. The FxCycle™ Violet stain uses the popular violet laser line and preferentially stains dsDNA by associating with AT clusters in the minor groove. Binding of FxCycle™ Violet stain to dsDNA results in ~20-fold fluorescence enhancement. The FxCycle™ Far Red stain uses the common 633 or 635 red laser line, and labels RNA as well as dsDNA.
 
what they offer
  • cell cycle analysis using 405 nm or 633/635 nm excitation sources for more flexibility in experimental design
  • minimal compensation with 488 nm excitable dyes for simpler analysis
  • tight CVs for accurate results
  • packaged as solid dye for longer shelf life and value

FxCycle™ Violet stain
Fluorescence excitation and emission profiles of FxCycle™ Violet stain (A) and FxCycle™ Far Red stain (B) bound to dsDNA.

FxCycle™ Violet and FxCycle™ Far Red stains
DNA content analysis using FxCycle™ Violet and FxCycle™ Far Red stains. (A) Histogram of HL-60 promyeloblast cells stained with FxCycle™ Violet stain showing DNA content distribution. Violet 405 nm excitation was used with a 450/50 bandpass filter. (B) Histogram of TF-1 erythroblast cells stained with FxCycle™ Far Red stain showing DNA content distribution. Red 633 nm excitation was used with a 660/20 bandpass filter. In both plots, G0/G1 and G2/M phase histogram peaks are separated by the S phase distribution.

Product Quantity Cat. no.
FxCycle™ Violet stain1 kit F10347
FxCycle™ Far Red stain1 kit F10348

Compensation control beads for LIVE/DEAD® fixable dead cell stains

what it is

The ArC™ Bead Kit provides a consistent, accurate and simple-to-use technique for the setting of flow cytometry compensation, and is specifically optimized for use with all of the LIVE/DEAD® Fixable Dead Cell Stain Kits.

how it works
Accurate compensation is crucial for effective multicolor analysis in flow cytometry. The LIVE/DEAD® Fixable Dead Cell Stain Kits employ an amine-reactive fluorescent dye to evaluate mammalian cell viability by flow cytometry. To set compensation, Arc™ reactive beads are incubated with LIVE/DEAD® fixable dead cell stain, washed in staining buffer, a drop of negative control beads is added, and the beads are resuspended and analyzed by flow cytometry. The two components provide distinct positive and negative populations of beads that can be used to set compensation. The difference in intensity between the bead populations is typically greater than 50-fold.

what it offers
  • fast and simple bead-based compensation for amine-reactive dyes
  • accurate and consistent results
  • easy combination with the AbC™ Anti-Mouse Bead Kit for multicolor immunophenotyping
  • does not require treating cells for use in compensation setting


Compensation using the ArC™ Bead Kit
Compensation using the ArC™ Bead Kit. Staining profile of the ArC™ Bead Kit components with two LIVE/DEAD® Fixable Dead Cell Stain Kits. (A) LIVE/DEAD® Fixable Blue dye–stained beads were analyzed with UV excitation; emission was collected with a 450/50 nm bandpass filter. (B) LIVE/DEAD® Fixable Violet dye–stained beads were analyzed with 405 nm excitation; emission was collected with a 450/50 nm bandpass filter.

Product Quantity Cat. no.
ArC™ Bead Kit1 kit A10346

Combine Organelle Lights™ and pHrodo™ reagents to visualize endocytosis and protein trafficking in real-time

what it is
A powerful combination of fluorescent protein–based Organelle Lights™ reagents and pH-sensitive pHrodo™ reagent–labeled proteins facilitates visualization of protein internalization and trafficking in live cells.   

how it works
The pHrodo™ dye is a novel, red-emission, pH-sensitive dye that dramatically increases fluorescence as the pH of its surroundings becomes more acidic, making it an ideal tool to study endocytosis and its regulation by drugs and/or environmental factors. Organelle Lights™ reagents are ready-to-use fusion constructs prepackaged in baculovirus particles for highly efficient cellular delivery. Conjugates of the protein of interest (e.g., a receptor) are prepared with the amine-reactive pHrodo™ succinimidyl ester. The lysosomes or endosomes are labeled with the appropriate GFP-based Organelle Lights™ reagent, and overnight incubation allows expression of the targeted fluorescent protein. GFP is the recommended fluorescent protein, as it can be easily distinguished from the red fluorescence of the pHrodo™ dye. Once the cells are ready, add the pHrodo™ conjugate and incubate while imaging the conjugate as it travels through this dynamic cellular pathway.

what it offers

  • real-time analysis—pHrodo™ and Organelle Lights™ reagents are live-cell compatible
  • content-rich results—pHrodo™ and Organelle Lights™ reagents are amenable to formaldehyde-based fixation and can be multiplexed with blue- and far-red–fluorescent dyes or labeled antibodies


Organelle Lights™ Endosome-GFP HeLa cells transduced with Organelle Lights™ Endosome-GFP. The following day, cells were washed and then incubated with 10 mg/mL pHrodo™ dextran and 1mg/mL Hoechst 33342 for 5 min. It is possible to visualize endocytosis of pHrodo™ by colocalization of Organelle Lights™ Endosome-GFP and pHrodo™ dextran. Cells were imaged on a Delta Vision Core microscope using standard DAPI/FITC/TRITC/Cy®5 filters.

 

Product Quantity Cat. no.
Organelle Lights™ Endosomes-GFP1 kitO10104
Organelle Lights™ Lysosomes-GFP1 kitO36228
pHrodo™, succinimidyl ester1 mgP36600


Learn more about Organelle Lights™ reagents.
Learn more about pHrodo™ reagents.

New validated primary antibodies for oncology, neurobiology, and immunology research

what they are
This month we are launching a diverse array of new cell signaling antibodies covering a broad spectrum of research areas, including oncology, neurobiology, immunology, and DNA modification. These antibodies are highly specific and validated with multiple applications using various species. The high quality of these antibodies is assured by ISO certified manufacturing techniques, and they are guaranteed to work when used as described. 

how they work
We are actively expanding our collection of primary antibodies to enable analysis of key cell signaling targets. These new antibodies include extracellular antibodies against the cytokines IL-23 and IL-17, neuroscience antibodies against Dopamine 1A, anti-acelated lysine antibody to detect histone/transcription factor modification, and a phosphospecific antibody against the tumor suppressor, retinoblastoma protein (Rb).

what they offer

  • confidence— all phosphorylation site specificities have been verified with peptide competition
  • performance—robust antibodies validated for multiple applications
  • selection—large menu of unique specificities, with wide range of targets and modification sites

 

RB [pS612], rabbit polyclonal antibody RB [pS612], rabbit polyclonal antibody: western blot and peptide competition. Extracts of Jurkat cells in high growth phase were resolved by SDS-PAGE and transferred to PVDF. The membrane was blocked with 5% BSA-TBST at room temperature for 1 hr, then incubated with RB [pS612] for 2 hr at room temperature in 3% BSA-TBST, following prior incubation with: no peptide (lane 1), a generic phosphoserine-containing (PS) peptide (lane 2), the non-phosphopeptide (NP) corresponding to the phosphopeptide immunogen (lane 3), or the phosphopeptide (P) immunogen (lane 4). After washing, the membrane was incubated with goat F(ab')2 anti–rabbit IgG alkaline phosphatase (Cat. no. ALI4405) and signals were detected using the Tropix® Western-Star™ detection method. The data show that only the phosphopeptide corresponding to Rb [pS612] blocks the antibody signal, demonstrating the specificity of the antibody.

 

Product Quantity Applications* Host (reactive
species)
Cat. no.
Mouse anti-IL-17 (human)500 µgELISAMs (Ch, Hu, Mk)433240
Mouse anti-IL-23 (human)500 µgELISAMs (Ch, Hr, Hu, Mk, Rt)433260
Mouse anti-Dopamine 1A receptor (rat)100 µgWB, IHCMs (Ms, Hr, Rt, Sw)434010
Mouse anti-Acetylated lysine100 µgWBVarious434020
RB [PS612] pAB10 blotsWBRb (Ck, Hu, Ms, Rt)44572G
* IHC = immunohistochemistry, WB = western blot.
† Ch = chimpanzee, Ck = chicken, Hr = horse, Hu = human, Mk = monkey, Ms = mouse, Rb = rabbit, Rt = rat, Sw = swine.


See a list of additional phosphorylation site–specific antibodies.
To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.

Fluorescence imaging 

Fluorescent probes for plant imaging

We’ve summarized a collection of literature and reagents to help you design experiments for your plant research. Organized by structural and functional application, you can easily find the fluorescent probes you need and identify published studies citing the use of these imaging reagents.

Download the plant imaging reference.


Fluorescence technologies for cell health and toxicity in high-content imaging and analysis

what they are

We offer a comprehensive set of solutions for determining cell health and cytotoxicity in cell-based assays on automated imaging and high-content screening (HCS) platforms.

how they work
HCS cell health and cytotoxicity assays use a wide variety of fluorescent probes to enable the fixed end-point detection of apoptotic, genotoxic, mitotoxic, and cytotoxic effects of drugs. The assays are compatible with detergent permeabilization and formaldehyde-based fixation, enabling antibody detection of on-target effects in the same cells. With these easy-to-use tools, simultaneous interrogation of multiple aspects of cytotoxicity and cell health is possible, allowing generation of high-content results needed to make the next critical decision in the process of drug discovery and development.

what they offer
The HCS cell health and cytotoxicity assays enable critical insights into various aspects of cell health:

  • apoptosis
  • cell proliferation
  • cell viability
  • DNA damage
  • mitochondrial health
  • mitotic index
  • nascent protein synthesis
  • phospholipidosis and steatosis


Detection of protein synthesis inhibition using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay. (A) Dose response curves, performed in duplicate. U2OS cells were treated in L-methionine–free medium containing 50 μM Click-iT® AHA and cycloheximide (85 pM–5 μM) for 30 min. Images show untreated cells (B) and cells treated with 150 μM cycloheximide (C).

Product Quantity Cat. no.
Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay, 2-plate size1 eachC10289
HCS DNA Damage Kit, 2-plate size1 eachH10292
HCS LipidTOX™ Green phospholipidosis detection reagent, 1000X aqueous solution, 10-plate size1 eachH34350
HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit, 2-plate size1 kitH34157
HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit, 10-plate size1 kitH34158
HCS LipidTOX™ Red phospholipidosis detection reagent, 1000X aqueous solution,10-plate size1 eachH34351
HCS LipidTOX™ Green neutral lipid stain, solution in DMSO1 eachH34475
HCS LipidTOX™ Red neutral lipid stain, solution in DMSO1 eachH34476
HCS LipidTOX™ Deep Red neutral lipid stain, solution in DMSO1 eachH34477
HCS LIVE/DEAD® Green Kit, 2-plate size1 eachH10290
HCS Mitochondrial Health Kit, 2-plate size1 eachH10295
HCS Mitotic Index Kit, 2-plate size1 eachH10293
Image-iT® DEAD Green™ viability stain, 1 mM solution in DMSO25 μLI10291


Learn more about HCS cell health and cytotoxicity assays at www.lifetech.com/hcs.

 

Experience the best—Alexa Fluor® dyes

From blue to red and into the near infrared, the Alexa Fluor® series of dyes provides many options for multicolor detection. Conjugates made with Alexa Fluor® dyes exhibit more intense fluorescence than other spectrally similar conjugates and are more photostable than most other fluorescent conjugates, allowing more time for image capture.

The Alexa Fluor® dyes remain the best and brightest conjugates we have ever tested. Recently, several new contenders have become available, most making claims of superior performance both in brightness and photostability. To investigate these claims, we compared all green-fluorescent secondary antibody conjugates available at the time of testing.

For more information about the Alexa Fluor® dye series, visit Alexa Fluor® Dyes.

Multicolor fluorescence  Multicolor fluorescence image of mouse testis cross section. Spermatozoa were detected using the green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (Cat. no. L21409). Actin filaments were visualized using red-fluorescent Alexa Fluor® 568 phalloidin (Cat. no. A12380). Nuclei were stained with blue-fluorescent DAPI (Cat. no. D1306, D3571, D21490).

 

A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.
Miksa, M. et al. (2009) J Immunological Methods 342:71–77.

How can we accurately assay the phagocytosis of apoptotic cells? Apoptosis comprises a multitude of highly regulated pathways through which an organism rids itself of unwanted cells without self injury, culminating in the phagocytic removal of post-apoptotic cellular debris by specialized immune cells such as macrophages and neutrophils. Errors (either in-born or acquired) in the machinery behind the phagocytosis of apoptotic cells are implicated in a number of medically important conditions; researchers need to be able to reliably assay phagocytosis in order to better understand these conditions. The phagocytic process can be viewed as occurring in distinct steps: binding of the unwanted object by the phagocytic immune cell, followed by the activation of signaling pathways, cytoskeletal rearrangements, and internalization. Methods that have been developed to assess phagocytosis by reporting the initial binding step can lead to overestimation of the extent of internalization under some circumstances. In their present 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. In their assay, target cells (apoptotic thymocytes) are loaded with the pH-sensitive fluorescent dye pHrodo™ succinimidyl ester (SE). Upon phagocytic engulfment by macrophages, the target cells are lysed and their contents encounter the acidic environment within the phagosome, causing a dramatic increase in pHrodo™ dye fluorescence. Through FACS flow cytometric analysis and direct observation through fluorescence microscopy, the authors used this assay to monitor the internalization of apoptotic thymocytes; their results showed excellent agreement with previously reported results. Additionally, they 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 in these cells upon addition of exogenous MFGE8. This study represents the first use of an assay of this type in eukaryotic cells; the assay could prove useful in future studies of the role of macrophages in apoptosis.

View bibliography reference.

Learn more about pHrodo™ reagents.
Human mesenchymal stem cell visualized using fluorescent protein–based Cellular Lights™ reagents. Stem cells were transduced with Cellular Lights™ Histone 2B-RFP (Cat. no. C10129) and Cellular Lights™ MAP4-GFP (Cat. no. C10105). Cells were imaged the following day using a DeltaVision® Core microscope with FITC/TRITC filter sets. Cytoskeletal dynamics and cell division in this living stem cell population were followed using time-lapse imaging. Image contributed by Nicholas Dolman and Scott Clarke, Life Technologies Corporation.
T-Cell Signaling Pathway New T cell receptor pathway web page
Whether your T cell receptor research requires basic research tools, cell-based assays, or comprehensive screening services, Invitrogen has solutions for you. Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate T cell receptor pathways—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 T cell receptor pathway research at www.invitrogen.com/tcellreceptor.

 Products in action videos:
Tools to make microscopy easier

See how hybridization covers and silicone isolators work.

HybriSlip™ hybridization covers are nuclease-free, ready-to-use and designed specifically for in situ hybridization. These hydrophobic coverslips do not require lengthy preparation or blocking procedures to prevent probe trapping or binding. HybriSlip covers are heat resistant and do not curl, even at high temperatures. They are ideal for denaturation steps or in situ PCR incubations.

Press-to-Seal™ silicone isolators are removable hydrophobic barriers that provide researchers with the ultimate in flexibility for isolating specimens. They may be used to isolate cells grown in culture dishes or to separate specimens on microscopy slides during staining procedures. The silicone material is autoclavable and adheres to any smooth surface.

 

High Content Screening - HCS 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
Accessorize Your Imaging—Encore Presentation March 31, 2009 10:00 a.m. PST
Fluorescence Immunolabeling Tips and Tricks April 14, 2009 10:00 a.m. PST


Missed our previous webinars? Find them at the links below:



Molecular Probes® The Handbook

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