Autophagy Assay Kits

Detects Autophagy in Living Cells
Autophagy is a conserved lysosomal recycling process by which cells break down their own components such as proteins, lipids, and carbohydrates. The process plays an important role in maintaining homeostasis and destroying intracellular pathogens. In addition, autophagy can be upregulated in times of starvation or stress to provide additional nutrients for the cell. Dysregulation of autophagy has implications for cancer, infection, and degenerative diseases.

Autophagy is a three-stage process. First, cytoplasmic components targeted for degradation are sequestered, resulting in the formation of the autophagosome. Next, the autophagosome fuses with the lysosome to form the autophagolysosome or autolysosome. Finally, degradation of the autophagosomal contents occurs.

Our Autophagy Assay, Red (cat# KF17373) enables researchers to detect and monitor the in vitro development of autophagy in living cells. The Autophagy Probe is cell-permeant and fluoresces red when inserted in the lipid membranes of autophagosomes and autolysosomes. Results can be read using a flow cytometer.

Figure. Flow Cytometry Results. Autophagy Assay Kit, Red was used to assess the induction of autophagy in Jurkat cells. Cells were either untreated (Black) or treated with 0.5 μM Rapamycin (Orange), 10 μM Chloroquine (Blue), or both 0.5 μM Rapamycin and 10 μM Chloroquine (Red) for 18 hours. After staining with Autophagy Probe, Red for 60 minutes, cells were washed and analyzed by flow cytometry (BD LSRFortessa Special Order flow cytometer equipped with a green/yellow laser (561 nm excitation) and a 610/20 emission filter). An overlay of the histograms is shown on the right. A table displaying the median fluorescence signal, % negative, and % positive cells is shown below. Treatment with Rapamycin or chloroquine increased the fluorescence signal detected compared to the untreated control. Combined treatment of rapamycin and chloroquine further increased the fluorescence signal detected. Data courtesy of Dr. Kristi Strandberg (ICT 228:37-40).

We have many options for detecting apoptosis, necrosis, autophagy, and cytotoxicity in living cells. 

Studying Apoptosis

By Cancer Researchers

Our Apoptosis Kits have proven rock solid in the hands of cancer researchers.

Here's data from a study of the impact of Vestibular schwannoma (VS) on hearing loss.

Images: Spiral Ganglion Cells labeled with our polycaspase kit stain to identify apoptotic cells.

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.

FLICA™ in vitro Caspase Kits for Apoptosis Assays

Great solution for measuring neurotoxicity

Here's a recent pub confirming the capabilities of our FLICA™ in vitro Caspase Apoptosis Detection Kits: Minghai Zhou, Gregory Ottenberg, Gian Franco Sferrazza, and Corinne Ida Lasmézas Highly neurotoxic monomeric α-helical prion protein. PNAS 2012 109 (8) 3113-3118; published ahead of print February 7, 2012, doi:10.1073/pnas.1118090109. ...PFA and stained for caspases using FLICA polycaspase assay (Neuromics) according to the manufacturer's instructions. Caspases...assayed on live cells using the caspase- specific FLICA assay (Neuromics). Cells were photographed using a Nikon inverted epifluorescence...

Images: FLICA was used to assess cell death in primay rat hippocampal neurons.Cells were plated on 25-mm poly-l-lysine-coated coverslips at 300,000 cells per coverslip. Cells were used at 4 or 8 days in vitro. Composite imagae (A) 3 out of 4 cells are apoptotic (green). No cells were necrotic as both of the PI-positive cells were FLICA-positive; they had compromised membranes and were probably in the late stages of apoptosis rather than necrosis. (B) 3 Caspase-positive cells fluoresce green.

I would like to underscore that we have solutions to cover most your needs for Apoptosis and Cytoxicity Assays. I will continue to post updates on customer data and publications.

Great Apoptosis Video

Related Reagents:
Apoptosis Research Reagents-Apoptosis Categories-includes: detection kits, antibodies and proteins

Apoptosis Signaling-Visualization and Measurement.

Apoptosis-Oxidative Stress Research Reagents are widely used and frequently referenced in customer publications. We work hard to keep our fingers on the pulse of how they are utilized across the many research areas important to our customers and add new reagents based on evolving requirements.

I recently posted publications referencing our MitoPT^TM Kits for quantitating Tumor Apoptosis

New Pub referencing Polycaspase Assay Kit, green: L. Wei, D. Ding and R. Salvi. Salicylate-induced degeneration of cochlea spiral ganglion neurons-apoptosis signaling.
doi:10.1016/j.neuroscience.2010.03.015.

Images: Typical confocal photomicrographs of SGN stained with Polycaspase Assay Kit (green) and with an antibody against neuronal III ß-tubulin (red) to identify SGN. (A) In control cultures, most SGN have large, oval shaped soma and neurites extending from the soma; note absence of polycaspase labeling (green). (B) SGN treated for 3 h with 5 mM SS; polycaspase labeling was present on SGN with shrunken soma. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Related Reagents:

Polycaspase Assay Kit, green

Magic Red™ Real Time! Kits -Measure apoptosis in
whole living, intact cells - no lysis required

FLIVO™ Polycaspase Live!, in vivo Apoptosis Kits-New
-Designed for cancer and neurodegenerative disease applications.

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 protease activation in whole living cells.

MitoPT™ Kits -Quantitate mitochondrial
functionality and apoptosis

MitoPT for Studying Tumor Apoptosis

We value our partnership with ICT. They provide our customers with potent and research proven Apoptosis Kits and Methods. Here we feature publications referencing our MitoPT™ Kits. These Kits easily assess changes in mitochondrial membrane potential. Changes in mitochondrial membrane potential can correlate with cytochrome c release and the initiation of apoptosis.
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.

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.

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.

Link Between Inflammation and Tau Pathology in Alzheimer's Disease

Providing reagents for Alzheimer's Disease Researchers is a strategic focus area for us. This includes making a variety of Primary Neuronal Cultures available to these researchers.

We evolve the strategy based on Researcher input and how the cultures are being referenced in publications. This reference is especially satisfying because of the vangaurd findings:

Lisette T. Arnaud, Natura Myeku and Maria E. Figueiredo-Pereira. Proteasome–caspase–cathepsin sequence leading to tau pathology induced by prostaglandin J2 in neuronal cells. Journal of Neurochemistry. Volume 110 Issue 1, Pages 328 - 342.

"Our data suggest a potential sequence of events triggered by the neurotoxic product of inflammation PGJ2 leading to tau pathology. The accumulation of Ub proteins is an early response. If cells fail to overcome the toxic effects induced by PGJ2, including accumulation of Ub proteins, apoptosis kicks in triggering caspase activation and tau cleavage, the clearance of which by cathepsins could be compromised culminating in tau pathology. Our studies are the first to provide a mechanistic link between inflammation and tau pathology."

Featured Reagent:
E18 Primary Rat Cortical Neurons

Related Reagents:

STEMEZ(TM) hN2 Human Neurons Discovery Kit

Images: Neural phenotypes derived from hN2 cell lines. (A) Phase contrast image of differentiated culture. (B) Network including post-mitotic motoneurons (HB9). (C) Cholinergic neuron. (D) Tuj-1 positive cells that are DAT-positive (dopamine transporter; closed arrow) and DAT-negative (open arrow). (E) Gabaergic neurons, inset illustrates GABA in axon, but not the dendrites (arrow).

Tau (Tau 46) Mouse Antibody

Tau Chicken Antibody

Apoptosis Research Reagents

We will continue to post "whats new" in AD research and potential uses of our reagents.

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