Bright Minds, Bright Solutions

Fun Holiday Correlation: FLICA-Staining and Brining a Turkey

2011-12-21T10:38:00.000-06:00

Greetings to you in this festive holiday week! As I imagine to be true for labs and offices around the country, holiday traditions have been a lively point of conversation among ICT scientists, colleagues, and customers this month. In particular, the great tradition of preparing a holiday feast - with a turkey - has garnered a lot of attention:

The logistics of arranging dinner for 25 humans and 6+ four-legged friends.
"Yams" vs. "sweet potatoes."
To bag or not to bag.
How not to use a turkey fryer. [Have you seen William Shatner's video?]
And brining a turkey - "... wait, isn't brining for pickles?"

That last topic led me to a fun realization...

the working concept behind brining a turkey is similar to that of our FLICA caspase detection assay! Both procedures work by utilizing cell-permeant molecules and the principle of diffusion. (You don't end up with a fluorescent turkey.)

The purposes and results, of course, are quite different: one provides a moist turkey for your holiday feast (hopefully), while the latter provides an easily analyzed answer to an investigation of apoptosis (reliably!).

Of course, brining a turkey is more than diffusion (and as some claim, osmosis, to some extent); oh no, there is one more fabulous scientific principle at play: denaturing. The increase in the salt concentration in the meat denatures protein strands, which causes them to retain more water during cooking, resulting in a moist and flavorful meal.

In the case of probing in vitro cell samples with FLICA, the act of diffusion is the first step towards the desired result, enabling whole, living cell analysis. After the fluorescent FLICA probes are inside the cells, this caspase detection mechanism relies on other principles of molecular biology:

enzymatic affinities for particular peptide sequences (e.g. V-A-D is targeted by all caspases)
ability of our proprietary inhibitor reagents to form covalent bonds with the active sites of caspase enzymes
and again, diffusion of unbound reagent from the cell during wash steps

Wishing you all Joyful Holidays and a Prosperous New Year!

Fun sources:
http://gizmodo.com/5862312/brine-your-turkey-fool-its-science
http://science.kqed.org/quest/2009/11/25/the-science-behind-brining/
http://www.virtualweberbullet.com/brining.html

More information about our cell-permeant protease detection probes:
http://www.immunochemistry.com/products/fluorescent-detection-reagents-for-apoptosis-caspase-activity-mitochondrial-permeability.html
http://www.immunochemistry.com/products/detection-reagents/flica.html

Measuring Caspase-1 Activity

2011-11-02T08:48:00.000-05:00

Caspase-1 (ICE)

• Originally identified as Interleukin -1β Converting Enzyme or ICE
• Cleaves IL-1 beta and IL-18 into active forms
• Creation of the inflammasome during host responses to pathogens leads to the activation of caspase-1
• Active caspase-1 plays an antimicrobial role during infection, leading to pyroptosis
• Inflammation-related disease models have illustrated a role for caspase-1 in asthma, rheumatoid arthritis, multiple sclerosis and other disorders

Activated THP-1 cells show caspase-1 (ICE) activity with the green fluorescent FAM-FLICA Caspase-1 Detection Assay

FAM-FLICA™ Caspase-1 Assay
This in vitro assay employs a fluorescent green inhibitor probe to detect active caspase-1 enzymes in living cells or tissue samples.
View assay details and data: http://www.immunochemistry.com/products/green-flica-caspase-1-detection-assay-kit.html

FAM-FLICA Caspase-1 Assay Kit

• Direct, fast labeling of active caspase-1

• Flexible analysis with standard fluorescence detection methods

• Green fluorescence allows for multiplexing with blue, orange, and red fluorescent dyes

Recent Citations

Our FLICA™ Caspase-1 Assay (catalog number 98, containing FAM-YVAD-FMK reagent) continues to be routinely referenced in customers' publications. Here are a few of the latest:

E. D. Murphey. Cecal Ligation and Puncture-Induced Impairment of Innate Immune Function Does Not Occur in the Absence of Caspase-1. J. Immunol. 2011; 187:905-910.

Mélanie Anne Hamon and Pascale Cossart. K+ efflux, is required for histone-H3 dephosphorylation by Listeria LLO and other pore forming toxins. Infect. Immun., Apr 2011; 10.1128/IAI.01243-10.

Norma Olivares-Zavaleta, Aaron Carmody, Ronald Messer, William M. Whitmire, and Harlan D. Caldwell. Chlamydia pneumoniae Inhibits Activated Human T Lymphocyte Proliferation by the Induction of Apoptotic and Pyroptotic Pathways. J. Immunol. 2011; 186:7120-7126.

Technical Tips for Measuring Caspase-1 with FLICA™

Create positive controls by inducing caspase-1 with a known inducing agent. Search publications for the best method of caspase-1 induction in your particular cell line. For example, we have used PMA and LPS with THP-1 cells to create known positives. Email us for more information.
Perform thorough wash steps with a centrifuge and, if necessary, a centrifuge plate adaptor. Passive washing with adherent cells is also possible by repeating the following steps 3x: aspirate the cell culture media, replace it with fresh media, and wait 10 minutes. However, some caspase-positive cells may have lost adherence and could be loose in the media; for this reason, the aspirated media must be collected and spun down for inclusion with the final analysis.

Coming soon: Red FLICA™ Caspase-1 Reagents
Perform multiplexing studies with green-labeled biomarkers via flow cytometry, fluorescence plate reader, and fluorescence microscopy.
Join our mailing list to be informed when this new product becomes available!

©2011 ImmunoChemistry Technologies, LLC. All Rights Reserved. | Bright Minds, Bright Solutions.

Nature Lab Offers 2011

2011-10-18T11:16:00.001-05:00

Each fall, the Nature Publishing Group assembles a group of exclusive research product offers from worldwide laboratory suppliers and distributes them as Nature Lab Offers. Look for your copy of 2011 Nature Lab Offers with the September and October U.S. editions of Nature and Nature Methods, or view Lab Offers online!

See your copy of Nature Lab Offers to learn about ICT's 2011 Lab Offer on ELISA Solutions™ for immunoassay development and in vitro diagnostic test manufacturing.

Build a Better Assay with ELISA Solutions™

· REDUCE BACKGROUND NOISE

· STABILIZE ENZYME CONJUGATES

· INCREASE SHELF LIFE OF COATED PLATES

· MINIMIZE MATRIX EFFECTS

Optimize your assay from start to finish with ICT's ELISA diluents, buffers, stabilizing diluents, solutions, and ELISA/EIA accessories for immunoassay development and in vitro diagnostic test manufacturing.

Antibody Isotyping Strips	HRP Anti-IgG Secondary Antibodies
Assay Diluents	HRP Maleimide, Conjugation-Ready
Blocking Buffers	Phosphate Buffered Saline
Coating Buffers	Plates and Accessories
Conjugate Stabilizer Diluents	Sample Diluents
ELISA Development Template Kits	Stop Solutions
ELISA Wash Buffer	Substrate Solutions, Colorimetric

Tools for Fluorescence-based Apoptosis and Caspase Detection

2011-09-21T16:37:00.000-05:00

Apoptosis is an evolutionarily conserved form of cell suicide responsible for various biological processes including the normal turnover of cells and the proper functioning of the immune system. This process of shutting down old, diseased, or otherwise disfunctional cells is mediated by a cascade of proteolytic enzymes known as cysteine proteases, or caspases. Pro-apoptotic signals activate the enzymatic cascade resulting in the cleavage of protein substrates, leading to the disassembly of the cell (1-4). Caspases have been identified in organisms ranging from C. elegans to humans. Members of the
mammalian caspase family of cysteinyl aspartate-specific proteases play distinct roles in apoptosis and inflammation.

Detection of activated caspase enzymes has much utility in basic and applied life science research, including the study of and the development of therapies against many cellular pathologies such as cancer and neurodegenerative disease. Among the research tools available for the qualitative and quantitative analysis of apoptosis-related proteins, fluorescence-based assays are useful for their flexible methods of analysis and multiplexing capabilities.

FLICA™: Caspase Detection Assay Utilizing Fluorescent-Labeled Inhibitors
This fluorescence-based caspase detection assay utilizes a fluorescent inhibitor reagent that forms a covalent bond with active, intracellular caspase enzymes, preventing them from further catalysis and labeling them for fluorescence detection. The use of a blue nuclear dye, Hoechst 33342, and a red vital dye, Propidium Iodide, is optional to acquire additional information about nuclear morphology and cell membrane permeability (necrosis) in the assay.

Multiplexing with FLICA™: Propofol-treated hippocampal neurons exhibit cell death via caspase-3/7 activity, as detected by the FAM-FLICA Caspase 3/7 Assay Kit. Caspase-3/7-positive cells fluoresce green with the caspase probe FAM-DEVD-FMK while nuclear morphology is visualized with blue Hoechst 33342 DNA dye. Dying membrane-permeant cells fluoresce red with the vital dye Propidium Iodide. Data courtesy of Dr. Sibel Kahraman, U-MD School of Medicine.

ImmunoChemistry Technologies (ICT) developed this in vitro assay in the late 1990s, expanding upon the line with additional caspase targets and fluorophore labels. The assays for poly-caspases and for effector caspases-3-and-7 are sought after for their utility as overall indicators of apoptosis. An increasing number of citations for ICT’s protease detection products appear each month in peer-reviewed publications as research continues to discover new chemotherapeutic methods to accelerate cancer cell death or slow the progression of neurodegenerative processes.

Assess apoptosis and necrosis in cultured cells with FLICA™

Sensitive and early indicator of the initiation of the apoptotic process
Distinguish apoptosis from necrosis
Flexible analysis methods
Green and red probes available
Detect poly caspases or individual caspase enzymes

Caspase Target; Use	Fluorescence	Kit
Poly Caspases; apoptosis	FAM (green) SR (red)	FAM-FLICA™ Poly Caspases Assay Kit SR-FLICA™ Poly Caspases Assay Kit
Caspase 1 (ICE); pyroptosis, inflammation	FAM (green)	FAM-FLICA™ Caspase 1 Assay Kit
Effector Caspases 3/7; apoptosis	FAM (green) SR (red)	FAM-FLICA™ Caspases 3/7 Assay Kit SR-FLICA™ Caspases 3/7 Assay Kit
Initiator Caspase 8; extrinsic apoptosis pathway	FAM (green)	FAM-FLICA™ Caspase 8 Assay Kit
Initiator Caspase 9; intrinsic apoptosis pathway	FAM (green) SR (red)	FAM-FLICA™ Caspase 9 Assay Kit SR-FLICA™ Caspase 9 Assay Kit

See the full kit listing, data, and citations at www.immunochemistry.com/products/detection-reagents/flica.html.

References:
1. Slee, E. A., C. Adrain, and S. J. Maritin. (1999) Serial Killers: ordering caspase activation events in apoptosis. Cell Death Differ. 6:1067-1074.
2. Earnshaw, W.C., Martins, L.M., and Kaufmann, S.H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Ann. Rev. Biochem. 68:383-424.
3. Hengartner, M.O. (2000) The biochemistry of apoptosis. Nature 407:770-816.
4. Degterev, A., Boyce, M., and Yuan, J. (2003) A decade of caspases. Oncogene 22:8543-8567.

Featured Summer Citations for Apoptosis, Caspase, and Cathepsin Detection

2011-08-31T12:27:00.000-05:00

Our fluorescent protease detection probes continue to be routinely referenced in customers' publications. Here are a few of the latest:

Neuronal toxicity assessed by SR-FLIVO™: Nuclear translocation of AMPK-α1 potentiates striatal neurodegeneration in Huntington’s disease
Tz-Chuen Ju, Hui-Mei Chen, Jiun-Tsai Lin, Ching-Pang Chang, Wei-Cheng Chang, Jheng-Jie Kang, Cheng-Pu Sun, Mi-Hua Tao, Pang-Hsien Tu, Chen Chang, Dennis W. Dickson, and Yijuang Chern
J. Cell Biol., Jul 2011; 194: 209-227. [Abstract]

Apoptosis detected with FAM-FLICA™ Poly Caspases Reagent: Asthmatic Airway Epithelium Is Intrinsically Inflammatory and Mitotically Dyssynchronous
Robert J. Freishtat, Alan M. Watson, Angela S. Benton, Sabah F. Iqbal, Dinesh K. Pillai, Mary C. Rose, and Eric P. Hoffman
Am. J. Respir. Cell Mol. Biol., Jun 2011; 44: 863-869. [Abstract]

Cathepsin activity detected with Magic Red™: Lysosomal Proteolysis Inhibition Selectively Disrupts Axonal Transport of Degradative Organelles and Causes an Alzheimer's-Like Axonal Dystrophy
Sooyeon Lee, Yutaka Sato, and Ralph A. Nixon
J. Neurosci., May 2011; 31: 7817-7830. [Abstract]

Image Apoptosis In Vivo! NIR-FLIVO Tracers Now Available

2011-08-30T14:54:00.003-05:00

ImmunoChemistry Technologies, LLC is pleased to announce the launch of two new tracers for non-invasive in vivo optical imaging of apoptosis. NIR-FLIVO™ 690 and NIR-FLIVO™ 747 in vivo apoptosis tracers and free dye control reagents are now available from ImmunoChemistry Technologies at introductory pricing.

Learn more about the new NIR-FLIVO in vivo apoptosis tracers at
www.immunochemistry.com/new-near-infrared-tracers-non-invasive-vivo-imaging-apoptosis.

The FLIVO™ product line utilizes proprietary in vivo tracer technology for apoptosis detection in live animal models. The non-toxic, cell-permeant FLIVO™ reagents specifically label apoptotic cells in vivo for analysis with fluorescence detection methods. These tracers, aka fluorescent probes, have been successfully used in a range of animal models to assess apoptosis as a result of a treatment or biological condition.

The new NIR-FLIVO™ apoptosis tracers utilize the near-infrared fluorescent labels DyLight® 690 and DyLight® 747 for noninvasive detection with whole animal imaging systems. Additionally, visible FLIVO™ tracers are available with carboxyfluorescein (FAM, green) or sulforhodamine-B (SR, red) labels for ex vivo optical imaging or flow cytometric analysis of histological samples.

These first-generation FLIVO tracers (FAM-FLIVO, SR-FLIVO) have proven to be useful for in vivo labeling of apoptotic cells, such as detecting apoptosis in vivo in ischemic hepatic tissue and in vivo labeling of cutaneous or shallow subcutaneous tumor growth entities. These shorter spectral-wavelength dyes tend to perform optimally when using ex vivo analysis methods.

Read more at www.immunochemistry.com/products/fluorescent-detection-reagents-for-apoptosis-and-protease-detection/in-vivo-apoptosis.html.

Join our mailing list to receive future product release updates.

FLIVO™ is a trademark of ImmunoChemistry Technologies, LLC.
DyLight® is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.

Federal Qualified Therapeutic Discovery Project Grants Awarded to ICT

2010-11-10T17:36:00.003-06:00

Bloomington, MN, November 1, 2010 - ImmunoChemistry Technologies announced that they will receive two Qualifying Therapeutic Discovery Project Grants from the IRS in conjunction with the U.S. Department of Health and Human Services.

Near Infrared Tracers For Imaging Apoptosis In Vivo ($243,031.30)
ICT's technology utilizes cell membrane permeable fluorescent dyes, conjugated to a pan caspase enzyme inhibitor, to non-invasively image apoptosis in vivo. In this project, we are the first to develop fluorophores in the near-infrared range that are cell membrane permeable to create tracers for optical imaging of apoptosis.

Having recently validated the ability of these near-infrared tracers to detect apoptosis in vitro, we have begun testing these tracers in mouse models of breast and prostate cancer and glioma. To date, the data demonstrate: a) the basal level of apoptosis present within the tumor, b) enhanced levels of apoptosis following treatment with anti-cancer therapy, c) specificity of uptake of the enzyme inhibitor as opposed to the dye, and d) the pharmacodynamics and kinetics of tracer uptake and clearance. These data are to be repeated and extended to other cancer therapy models as well as other animal models of disease.

We anticipate launching these new near-infrared apoptosis detection reagents into the marketplace before the end of 2010. These new near-infrared tracers will join our current FLIVO™ and FLICA™ product lines that contain fluorescein and sulforhodamine-conjugated apoptosis detection reagents.

Moreover, there are some applications to human diagnostics and therapeutics that may benefit from the use of these novel near-infrared tracers, namely in ophthalmology, cardiology, dermatology, and melanoma. We therefore intend to pursue these reagents further into the pre-clinical and then clinical phases of approval by the FDA.

MRI of Apoptosis Using New Tracers Specific for Caspase Activity ($111,386.50)
ICT's technology utilizes cell membrane permeable reporter molecules, conjugated to a pan-caspase enzyme inhibitor, to detect apoptosis. In this project, we are developing novel 19-F magnetic-resonance-based tracers for imaging (MRI) apoptosis. We have recently validated the ability of two 19-F MRI candidate tracers to detect apoptosis in vitro. We have six more candidate tracers that are in the pipeline to be synthesized.

When the tracers have all been validated (or excluded) for their ability to bind caspase enzymes in vitro, we will select the MRI tracers that exhibit the best binding characteristics for further testing in experimental animals. Accordingly, we will then evaluate: a) the required chemistry to create the tracers in quantities and purity necessary for commercial production, b) the basal levels of apoptosis in experimental animal models of disease and the enhanced levels of apoptosis exhibited following treatment, c) the dosing requirements and toxicity of the candidate tracers in experimental animals, and d) the pharmacodynamics and kinetics of clearance of the MRI tracer's uptake and clearance. We anticipate that our novel MRI tracer(s) will be launched into the research-use only market sometime in 2012.

More pertinent, however, are the applications to human diagnostics and therapeutics that will benefit from the use of this novel MRI tracer. The ability to detect apoptotic cells in human patients using MRI impels us to pursue these reagents further into the pre-clinical and then clinical phases of FDA approval.

Learn more about ICT's research at http://www.immunochemistry.com/research.

©2010 ImmunoChemistry Technologies | Bright Minds, Bright Solutions™

New Products: Affipure Goat HRP-Anti-IgG Secondaries

2010-10-29T14:16:00.002-05:00

Affipure Goat HRP-Anti-IgG Secondaries for all ELISA formats and Western blotting are now available from ICT:

HRP Goat anti-Human IgG Fc
HRP Goat anti-Rabbit IgG Fc
HRP Goat anti-Mouse IgG Fc

The IgG components of these conjugates are specific for normal IgG-Fc of their target species. Each vial contains 1.0 mg of Affinity Purified Goat IgG conjugated to 2-4 HRP enzymes per IgG molecule. The conjugate is dissolved in a Tris based conjugate stabilization buffer containing 50 ppm ProClin 300 anti-microbial agent.

Check our New Products section periodically for more product releases and updates from ICT!

ImmunoChemistry Technologies offers a specialty line of ELISA products for custom immunoassay development and manufacturing. All of our ELISA Solutions™ work together to build an accurate and sensitive immunoassay with minimal background noise. ICT’s coating and blocking buffers stabilize the plate; our sample and assay diluents help generate an accurate standard curve. We also offer conjugate stabilizers, substrates, and more. Optimize your assay from start to finish with ELISA Solutions™.

ICT's new online home has launched!

2010-10-15T17:32:00.001-05:00

On September 17, 2010, ICT launched a completely new design on our website, immunochemistry.com. We added new features, such as a document library, instant credit card processing for product orders, and in-depth information about our assay development and custom laboratory services.

Expect to see more changes as we make improvements and add new products. Here's a humorous breakdown of what Erin and Sally did to get the website working. Good things are happening at ICT in 2010!

New publications employ use of green FAM FLICA caspase assays for apoptosis detection

2010-04-30T13:03:00.004-05:00

Following a low pH and H2O2 treatment, keratoconus fibroblast cultures (right) exhibit higher levels of caspase-3 activity than normal cultures (left) via staining with FAM FLICA caspase 3/7 detection reagent FAM-DEVD-FMK. Data courtesy of Drs. Kenney and Luthra, University of California Irvine Medical Center.

Globular Adiponectin as a Complete Mesoangioblast Regulator: Role in Proliferation, Survival, Motility, and Skeletal Muscle Differentiation
Fiaschi T, Tedesco FS, Giannoni E, Diaz-Manera J, Parri M, Cossu G, and Chiarugi P. Mol. Biol. Cell, Mar 2010; 21: 848 - 859.
...carboxyfluorescein FLICA (fluorescent-labeled inhibitor of caspases) apoptosis detection kit was from ImmunoChemistry Technologies (Bloomington... ...cells undergoing apoptosis was assayed by...FLICA apoptosis detection kit according to...manufacturer's protocol... Briefly, nonpermeabilized D16 cells were washed...

The Endoplasmic Reticulum Stress-Inducible Protein, Herp, Is a Potential Triggering Antigen for Anti-DNA Response
Hirabayashi Y, Oka Y, Ikeda T, Fujii H, Ishii T, Sasaki T, and Harigae H. J. Immunol., Mar 2010; 184: 3276 – 3283.
...To detect apoptosis, caspase activity was visualized as a green color using an FAM FLICA poly-caspase assay kit (Immunochemistry Technologies, Bloomington, MN)…

CD56highCD16–CD62L– NK Cells Accumulate in Allergic Contact Dermatitis and Contribute to the Expression of Allergic Responses
Carbone T, Nasorri F, Pennino D, Eyerich K, Foerster S, Loredana Cifaldi L, Traidl-Hoffman C, Behrendt H, and Cavani A. J. Immunol., Jan 2010; 184: 1102 - 1110.
...prior to NK cell exposure. Induction of keratinocyte apoptosis was determined using the caspases 3 and 7 FLICA kit (ImmunoChemistry Technologies, Bloomington, MN) according to the manufacturers protocol, and evaluated by FACS. ELISA Supernatants of NK cells...

MitoPT JC-1 assay recently cited in multiple in vitro cancer therapeutic studies

2010-03-10T15:19:00.003-06:00

A431 cells, treated with predetermined IC50 concentration of novel anticancer agents, fluoresce green and orange-red with MitoPT JC-1. Data courtesy of Zayas/ Carro, Universidad Metropolitana.

Anticancer Effects of Alpinia pricei Hayata Roots.
CL Hsu, YS Yu, GC Yen. J. Agric. Food Chem., Jan 2010, 58 (4), pp 2201–2208.
... Mitochondrial Membrane Potential (ΔΨ m ) Analysis… The mitochondrial membrane potential was determined using a MitoPT 100 test kit (Immunochemistry Technologies, LLC, Bloomington, MN). JC-1 is a cationic dye that exhibits ...

Anticancer Effects of Flavonoid Derivatives Isolated from Millettia reticulata Benth in SK-Hep-1 Human Hepatocellular Carcinoma Cells.
SC Fang, CL Hsu, HT Lin, GC Yen. J. Agric. Food Chem., Jan 2010, 58 (2), pp 814–820.
... About 1 × 10^4 events were acquired and analyzed using CELL Quest software. Mitochondrial Membrane Potential (ΔΨ m ) Analysis… Mitochondrial membrane potential was determined using the MitoPT 100 test kit (Immunochemistry Technologies, LLC). ...

Mechanisms of Apoptotic Effects Induced by Resveratrol, Dibenzoylmethane, and Their Analogues on Human Lung Carcinoma Cells.
CJ Weng, YT Yang, CT Ho, GC Yen. J. Agric. Food Chem., Jun 2009; 57 (12), pp 5235–5243.
... Collapse of Mitochondrial Membrane Potential (ΔΨm) by Resveratrol, MR-3, and HMDB… The mitochondrial permeability transition is an important step in the induction of cellular apoptosis. The loss of mitochondrial membrane potential is a hallmark for apoptosis... The mitochondrial permeability transition detection kit (MitoPT) was purchased from Immunochemistry Technologies…

Recent Publications Highlight the Use of FLIVO to Detect Apoptotic Dendritic Cells in vivo

2010-02-04T13:26:00.001-06:00

CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3β
Cristina Escribano, Cristina Delgado-Martín, and José Luis Rodríguez-Fernández. J. Immunol., Nov 2009; 183: 6282 – 6295.
...Sulforhodamine B (SR)-FLIVO (SR, abs 565 nm, em 600 nm), a form of FLIVO conjugated to sulforhodamine B, was obtained from Immunochemistry Technologies... SR-FLIVO, an agent that selectively stains apoptotic (cells) in vivo...

Immunological synapse formation inhibits, via NF-kappaB and FOXO1, the apoptosis of dendritic cells.
Riol-Blanco L, Delgado-Martín C, Sánchez-Sánchez N, Alonso-C LM, Gutiérrez-López MD, Del Hoyo GM, Navarro J, Sánchez-Madrid F, Cabañas C, Sánchez-Mateos P, Rodríguez-Fernández JL. Nat Immunol., 2009 Jul 10(7):753-60. Epub 2009 Jun 7.
... To identify apoptotic DCs inside the lymph node, we used FLIVO. ... mice were injected intravenously with sulforhodamine-conjugated FLIVO ... λem > 600 nm)—were obtained from Immunochemistry Technologies... Using FLIVO, we analyzed whether IS formation was able to protect DCs from becoming apoptotic in the lymph node (Fig. 3a)...

A protocol to detect apoptotic dendritic cells in murine lymph nodes using multiphoton microscopy.
Delgado-Martín C, Riol-Blanco L, Alonso-C L M, Rodríguez-Fernández J L. Nature Protocols. DOI: 10.1038/nprot.2009.133. June 2009.
... In non-apoptotic DCs, which lack caspases, FLIVO reagents are not retained and (diffuse) out. However, in apoptotic DCs they form covalent bonds with ... λem >600 nm) (Cat No 983)(Immunochemistry Technologies, LLC)... In studies to ascertain whether a treatment increases or reduces ... The method is also useful to analyse the apoptosis of T cells7 or any other cell type able to migrate ...

Recent Magic Red and FLICA Citations

2009-11-02T14:25:00.000-06:00

Magic Red
Xiang Shen, Jeong-Seok Park, Ye Qiu, Joel Sugar, and Beatrice Y J T Yue. Effects of Sp1 overexpression on cultured human corneal stromal cells. Genes Cells, Oct 2009; 14: 1133 – 1139.
...Cathepsin B activity The activity of cathepsin B in transfected cells was detected using the Magic Red cathepsin B kit (Immunochemistry Technologies, Bloomington, MN, USA). Ki-67 staining Transfected cells were fixed in cold methanol and permeabilized. After...

FLICA
Soumika Biswas, Kaushik Biswas, Amy Richmond, Jennifer Ko, Sankar Ghosh, Matthew Simmons, Patricia Rayman, Brian Rini, Inderbir Gill, Charles S. Tannenbaum, and James H. Finke. Elevated Levels of Select Gangliosides in T Cells from Renal Cell Carcinoma Patients Is Associated with T Cell Dysfunction. J. Immunol., Oct 2009; 183: 5050–5058.
...and GM2. To study activation of caspases, cells were stained with fluorochrome inhibitors of caspases (FLICA) from Immunochemistry Technologies for 1 h at 37C protected from light, followed by staining with hamster anti-human GM2 Ab. Induction of ROS was determined...

S. Grunewald, C. Kriegel, T. Baumann, H.-J. Glander, and U. Paasch. Interactions between apoptotic signal transduction and capacitation in human spermatozoa. Hum. Reprod., Sep 2009; 24: 2071–2078.
...the appropriate controls according to the kit instructions provided by the manufacturer (Carboxyfluorescein FLICA, Immunochemistry Technologies, Bloomington, MN, USA). A 150-fold stock solution of the inhibitor was prepared in DMSO and further diluted in phosphate...

FAM-FLICA Caspase-1
Unja Martin, John Scholler, Jesse Gurgel, Blair Renshaw, John E. Sims, and Christopher A. Gabel. Externalization of the Leaderless Cytokine IL-1F6 Occurs in Response to Lipopolysaccharide/ATP Activation of Transduced Bone Marrow Macrophages. J. Immunol., Sep 2009; 183: 4021–4030.
...anti-GFP mouse monoclonal from Clontech, and anti-His mouse monoclonal from Roche. Caspase-1 FLICA kit was from Immunochemistry Technologies. Recombinant mcsf-1 and CP-456773 were generated at Amgen. Isolation of murine macrophages C57BL/6 mice were...

Study describes role of Caspase-1 in infection of Chlamydia Trachomatis, use of FAM-YVAD-FMK cited

2009-10-09T11:30:00.001-05:00

In the well-crafted study of inflammasome induction following cervical epithelial cell infection, Abdul-Sater et. al. (Inflammasome-Dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia Trachomatis, J. Biol. Chem 284: 26789-26796, 2009) used ImmunoChemistry Technologies’ FLICA caspase-1 assay kit (catalog #98, FAM-YVAD-FMK) to quantify the percentage of cells containing active caspase-1 following C. Trachomatis infection of HeLa cells. The data show a significant level of caspase-1 activation within 24 hours post-infection evident in 50% of the cultured epithelial cells. The study goes further to illustrate the role of ROS production and K+ efflux in the activation of caspase-1. Specifically, infection by C. Trachomatis leads to a decrease in intracellular K+ levels that causes a rise in ROS production resulting in caspase-1 activation. Furthermore, the study illustrates how subsequent caspase-1 activation after C. Trachomatis infection enhances the infectivity of the pathogen.

Study Reveals Kinetics in Turnover of Young Rat Urothelium, Employing FAM-FLIVO™ for in vivo Apoptosis Detection

2009-10-06T14:03:00.000-05:00

The recently published study by Erman et. al. (Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development, J. Histochem. Cytochem. 57(8):721-730, 2009) used ImmunoChemistry Technologies’ FAM-FLIVO reagent (catalog #981) to observe the levels of apoptosis in urothelial cells of the young rat bladder. The study clearly demonstrates the kinetics in turnover of rat urothelium in the early postnatal period (birth to day 14) evidenced by apoptosis, autophagic cell death, and desquamation.

FLIVO and FLICA References

2009-09-21T11:27:00.000-05:00

FAM-FLIVO™
Andreja Erman, Dasa Zupancic, and Kristijan Jezernik, Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development. J. Histochem. Cytochem., Aug 2009; 57: 721 - 730.

FAM-FLICA™
Shira D. P. Rabin, Jared G. Flitton, and Donald R. Demuth, Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Induces Apoptosis in Nonproliferating Macrophages by a Phosphatase-Independent Mechanism. Infect. Immun., Aug 2009; 77: 3161 - 3169.

ImmunoChemistry Technologies Receives Fast Track Grant Award from National Institutes of Health

2009-09-11T18:42:00.000-05:00

Bloomington, Minn, September 11, 2009 – ImmunoChemistry Technologies, LLC, announced today that the National Cancer Institute (NCI) of the National Institutes of Health (NIH) has approved funding for Phase II of a fast track Phase I/II Small Business Innovation Research (SBIR) Grant, first awarded to the Company in September 2008. The award of over $640,000 is the second installment of the combined $850,000 grant to develop and test new reagents (FLIVO™) for fluorescence imaging of apoptotic tissues in various animal models of anti-cancer therapy. The work will be done in collaboration with the University of Minnesota.
Read more about our research and FLIVO™ on our website.

ImmunoChemistry Technologies Receives Funding from National Institutes of Health

2009-09-11T18:26:00.000-05:00

Bloomington, Minn, September 11, 2009 – ImmunoChemistry Technologies, LLC, announced today that the National Institute of Mental Health (NIMH) of the National Institutes of Health (NIH) has awarded the Company its 2nd year, $250,000 grant under its Small Business Technology Transfer Research (STTR) program to develop new reagents for future medical imaging of apoptotic tissues. The work will be done in collaboration with Vanderbilt University in Nashville, TN.
Read more about our research and our existing FLIVO™ tracers on our website.

Recent Citations using FLICA and Magic Red

2009-08-20T15:01:00.000-05:00

Cutting Edge: NF-B Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression
Franz G. Bauernfeind, Gabor Horvath, Andrea Stutz, Emad S. Alnemri, Kelly MacDonald, David Speert, Teresa Fernandes-Alnemri, Jianghong Wu, Brian G. Monks, Katherine A. Fitzgerald, Veit Hornung, and Eicke Latz
J. Immunol., Jul 2009; 183: 787 - 791.
...the caspase-1 carboxyfluorescein-YVAD-fluoromethylketone FLICA (fluorescent labeled inhibitors of caspases) kit (Immunochemistry Technologies) was used to stain active caspase-1. CD14-positive cells were analyzed for FLICA positivity by flow cytometry. The...

The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability
Shobbir Hussain, Sandra Blanco Benavente, Elisabete Nascimento, Ilaria Dragoni, Agata Kurowski, Astrid Gillich, Peter Humphreys, and Michaela Frye
J. Cell Biol., Jul 2009; 186: 27 - 40.
...fluorochrome inhibitor of caspases (FLICA) apoptosis detection kit was used as per the manufacturers instructions (Immunochemistry Technologies). FLICA probe, which covalently binds to activated caspases, was incubated with cells for 1 h, and cells were washed...

Sulforaphane targets pancreatic tumour-initiating cells by NF-B-induced antiapoptotic signalling
G Kallifatidis, V Rausch, B Baumann, A Apel, B M Beckermann, A Groth, J Mattern, Z Li, A Kolb, G Moldenhauer, P Altevogt, T Wirth, J Werner, P Schemmer, M W Büchler, A V Salnikov, and I Herr
Gut, Jul 2009; 58: 949 - 963.
...FAM-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (FLICA) was used according to the manufacturers protocol (Immunochemistry Technologies, Bloomington, Minnesota, USA). FLICA binds covalently to specific active caspases (thus the fluorochrome accumulates...

ATP-induced osteoclast function: the formation of sealing-zone like structure and the secretion of lytic granules via microtubule-deacetylation under the control of Syk
Ryoichi Hazama, Xiujuan Qu, Kunio Yokoyama, Chisato Tanaka, Emi Kinoshita, Jinsong He, Shusuke Takahashi, Kaoru Tohyama, Hirohei Yamamura, and Yumi Tohyama
Genes Cells, Jul 2009; 14: 871 - 884.
...Osteolytic granules were labeled with LysoTracker Red (Invitrogen) and Magic Red cathepsin K detection kit (Immunochemistry Technologies, Bloomington, MN, USA). Cathepsin K detection kit was also used to quantify the released enzyme activity in the...

Six New Citations using ICT's Kits

2009-07-30T18:04:00.000-05:00

Biological Significance of Prolactin in Gynecologic Cancers
Vera V. Levina, Brian Nolen, YunYun Su, Andrew K. Godwin, David Fishman, Jinsong Liu, Gil Mor, Larry G. Maxwell, Ronald B. Herberman, Miroslaw J. Szczepanski, Marta E. Szajnik, Elieser Gorelik, and Anna E. Lokshin
Cancer Res., Jun 2009; 69: 5226 – 5233
...and by analysis of caspase activation using Fluorescent Poly-Caspases FLICA Apoptosis Detection kit FAM-VAD-FMK (Immunochemistry Technologies) as previously described (17). Multiplex analysis of phosphoproteins. Analysis of phosphoproteins was performed... ...

Culling of Activated CD4 T Cells during Typhoid Is Driven by Salmonella Virulence Genes
Aparna Srinivasan, Minelva Nanton, Amanda Griffin, and Stephen J. McSorley
J. Immunol., Jun 2009; 182: 7838 – 7845
...was detected by flow cytometry using the FLICA apoptosis detection kit according to the manufacturer's instructions (Immunochemistry Technologies). Results Depletion of activated Salmonella-specific T cells in mice infected with live bacteria Several...

Deficiency of the DNA repair enzyme ATM in rheumatoid arthritis
Lan Shao, Hiroshi Fujii, Inés Colmegna, Hisashi Oishi, Jörg J. Goronzy, and Cornelia M. Weyand
J. Exp. Med., Jun 2009; 206: 1435 – 1449
...Particles to quantify cell numbers. Cell apoptosis was detected by caspases 3 and 7 SR FLICA kits (ImmunoChemistry Technologies), and rates of apoptotic cells were quantified through PI staining (Sigma-Aldrich). Comet assay. DNA damage was... ...

Farnesol-Induced Apoptosis in Candida albicans
Mark E. Shirtliff, Bastiaan P. Krom, Roelien A. M. Meijering, Brian M. Peters, Jingsong Zhu, Mark A. Scheper, Megan L. Harris, and Mary Ann Jabra-Rizk
Antimicrob. Agents Chemother., Jun 2009; 53: 2392 - 2401.
...mitochondrial permeability transition detection kit (Immunochemistry Technologies, LLC, Bloomington, MN) according to the manufacturers...microscopically using FLICA apoptosis detection kits (Immunochemistry Technologies, LLC) according to the manufacturer's recommendation... ...

Impairment of APE1 Function Enhances Cellular Sensitivity to Clinically Relevant Alkylators and Antimetabolites
Daniel R. McNeill, Wing Lam, Theodore L. DeWeese, Yung-Chi Cheng, and David M. Wilson, III
Mol. Cancer Res., Jun 2009; 7: 897 – 906
...red fluorescence above 600 nm. Apoptosis was measured using the poly-caspases FLICA apoptosis detection kit from Immunochemistry Technologies, LLC. The kit uses an inhibitor sequence of caspases (VAD, which reacts with all caspases) linked to a green (carboxyfluorescein... ...

Phenotypic and Functional Changes Induced in Hematopoietic Stem/Progenitor Cells After Gamma-Ray Radiation ExposureArthur J. Simonnet, Johnny Nehmé, Pierre Vaigot, Vilma Barroca, Philippe Leboulch, and Diana Tronik-Le Roux
Stem Cells, Jun 2009; 27: 1400 - 1409
...for another 15 minutes at 37C and immediately analyzed by flow cytometry. Apoptosis Assay A caspase 3/7 FLICA Kit, Immunochemistry Technologies, LLC, Bloomington, MN, http:www.immunochemistry.com, was used according to the manufacturer's protocol...

Welcome to ICT's Blog

2009-04-08T18:47:00.001-05:00

Based in Bloomington, Minnesota, ICT is one of the world's leading suppliers of unique in vivo and in vitro tools critical for discovering promising investigational medicines for treating life threatening illnesses.

Our fluorescent enzyme detection probes are used to assess apoptosis and cell death in cultured cells. ICT's specialty in vitro probes detect caspases, cathepsins, serine proteases, cholinesterase enzymes, and assess mitochondrial health. We also manufacture proprietary probes that can detect dead and dying cells in living organisms in vivo for research use. In the future, these probes may be used to better manage disease and personalize treatment. ICT's in vivo probes may be used to assess the efficacy of chemotherapy, to quantify neurodegeneration, and as an early detector of eye disease, among other applications.

ICT is also a contract research laboratory specializing in custom ELISA development, manufacturing, and testing services. As an out-growth of these services, ICT offers a specialty line of ELISA buffers and stabilizers to researchers developing in-house assays.