Fatty Acids and IBD and Colitis

The Dangers of a High Fatty Acid Diets
Our westernized high-fat diet (HFD) is associated with the development of inflammatory bowel disease (IBD), Colitis and other intestinal related diseases and cancers. Here one the root causes is identified to elevated production of pro-inflammatory cytokine-IL-1β by macrophages. It is suspected that HFD sets off an autoimmune like cascade.

This cascade is triggered by NLRP3 inflammasomes. Our Apoptosis Kit-Magic Red-Real Time Cathepsin B is used to demonstrate these inflammasomes are activated via Cathepsin B Release: Shengnan Zhao, Zizhen Gong1, Jiefei Zhou1, Chunyan Tian, Yanhong Gao, Congfeng Xu, Yingwei Chen, Wei Cai and Jin Wu1Deoxycholic Acid Triggers NLRP3 Inflammasome Activation and Aggravates DSS-Induced Colitis in Mice. Front. Immunol., 28 November 2016 | http://dx.doi.org/10.3389/fimmu.2016.00536 ...Lipopolysaccharide-primed J774A.1 cells were incubated with or without DCA (100 μM, 24 h); then, the cells were stained with Lyso Tracker Green DND-26 (Invitrogen) or magic red cathepsin B fluorogenic substrate z-Arg-Arg cresyl violet (Neuromics) for 1 h, followed by Hoechst staining for half an hour. Fluorogenic signals were captured by inverted fluorescence microscope (Leica)...

Image: Lipopolysaccharide-primed J774A.1 cells were incubated with or without DCA (100 μM, 24 h); then, the cells were stained with Lyso Tracker Green DND-26 (Invitrogen) or Magic RedTM Cathepsin B fluorogenic substrate z-Arg-Arg cresyl violet (Neuromics) for 1 h, followed by Hoechst staining for half an hour. Fluorogenic signals were captured by inverted fluorescence microscope (Leica). Front. Immunol., 28 November 2016 http://dx.doi.org/10.3389/fimmu.2016.00536

We will continue to post how our customers are using our Apoptosis Kits for their discoveries here.

Magic Red and Cancer Research

Measure Apoptosis in Real Time in Cancer Cells

Magic Red™ (MR) Kits measure apoptosis via active caspases and cathepsins in whole living, intact cells - no lysis required. As apoptosis progresses and caspase activity increases, the red fluorescent signal increases.

In this example, Magic Red is used to measure apoptosis in HeLa Cells after treatment with Artesunate (ART), an anti-malarial drug and potential chemotherapy agent.

Figure: ART activates lysosomal function. A, HeLa cells were first treated with ART (50 μM) for 12 h, followed by LTR labeling for 30 min, and further analyzed using confocal microscopy. B, HeLa cells were loaded with LTR following time course drug treatment, and fluorescence intensity of 10,000 cells/sample was measured by flow cytometry. C, HeLa cells were treated as indicated in A and then incubated with Magic Red cathepsin L and LTG for 30 min and observed under a confocal microscope. D, HepG2 cells were treated as in A and then labeled with LTR described earlier in A. E, HepG2 cells were treated as in A, followed by a Magic Red cathepsin B and L assay for 45 min. Fluorescence intensity of 10,000 cells/sample was measured using flow cytometry. F, HeLa cells were first loaded with DQ Red BSA for 1 h and then treated with ART (50 μM) for 18 h and incubated with LTG for 30 min. G, HeLa cells were treated with ART for 18 h and then immunostained for LAMP1. H, LAMP1 signals of 80 cells from three independent experiments were quantified by ImageJ (Student's t test, #, no significance). I, HeLa cells were treated with ART for the indicated time, and then Western blot was performed to detect the LAMP1 protein level. Scale bar, 10 μm. Error bars, S.D. doi: 10.1074/jbc.M114.564567

We have a variety of Apoptosis Kits for testing potential Chemotherapeutic Agents.

Detecting Apoptosis in Real Time

Kudos to Drs. Aaron Thomas and Carol Erickson for their novel use of our Magic Red™ Real Time! Apptosis Kits. Here's the related publication:

Aaron J. Thomas and Carol A. Erickson. FOXD3 regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism. Development, Apr 2009; doi:10.1242/dev.031989.
...Apoptosis was assayed using Magic Red Caspases 3&7 reagent (Neuromics)...

Related Reagents:
FLIVO™ Polycaspase Live!, in vivo Apoptosis Kits-New
FLICA™ in vitro Caspase Kits
Fast!-Use Caspase kits to quantitate apoptosis via active caspases in whole, living cells. These kits do not use ELISA or any antibodies for detection
FLISP™ Serine Protease Detection Kits
Measure chymotrypsin-like proteaseactivation in whole living cells.
MitoPT™ Kits
Quantitate mitochondrial functionality and apoptosis
Apoptosis Research Antibodies
Apoptosis Research Proteins