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. 

Co-Cultures and Tox Assays

Data End Points Differ
Our clients continue to use our cells and kits for co-culturing. The goal is to develop the most in vivo like results possible.

They find that single cell vs multi cell type co-cultures drive different data sets.

At each dose, the results vary and as the dose of Bis1 increases this variation increases.

We welcome your questions, input and feedback on our co-cultures. Pete Shuster, Owner, pshuster@neuromics.com or 612-801-1007.

hNP1 Neural Progenitors and HTS Tox Assays

Sensitivity of Neural Progenitor Cells to Chemical Induced Apoptosis

This article demonstrates the sensitivity of Neuromics' hNP1TM Neural Progenitors to chemically induced apoptosis: Ingrid Druwea, Theresa M. Freudenrich, Kathleen Wallace, Timothy J. Shafer, William R. Mundy. Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening. doi:10.1016/j.tox.2015.03.011.

Summary: The results demonstrate that, (1) all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format; (2) there were differences in the response of the rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro; and (3) proliferating neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery.

Here're more publications of referencing use of hNP1 Neural Progenitors:

Yuji Kaneko, Hideki Shojo, Jack Burns, Meaghan Staples, Naoki Tajiri, Cesar V. Borlongan, DJ-1 ameliorates ischemic cell death in vitro possibly via mitochondrial pathway, Neurobiology of Disease, Available online 21 September 2013, ISSN 0969-9961, http://dx.doi.org/10.1016/j.nbd.2013.09.007...Cell culture and oxygen-glucose deprivation (OGD) hNPCs were obtained from Neuromics...

Xiufang Guo, Severo Spradling, Maria Stancescu, Stephen Lambert, James J. Hickman. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials, In Press, Corrected Proof,Mar 2013.doi:10.1016/j.biomaterials.2013.02.061 ...hNP1, were obtained from Neuromics (Edina, Minnesota)...

Questions? Do not hesitate to contact me, Pete Shuster: pshuster@neuromics.com or direct phone: 612-801-1007.

Save 100 USD on our hN2™ Primary Neurons

Potent, Pure and Easy to Culture

Assay data continues to roll in. I am pleased by the high lot to lot consistencies of our hN2 Primary Human Neurons resulting in data reproducibility. Here're an examples of outgrowth/tox assays: In conjunction with Molecular Devices' ImageeXpress HCI platform, hN2 differentiated neuronal cells can be used to evaluate potential toxic effects of test compounds on nuerite outgrowth through visualizing cells stained with β-lll tubulin.

Figure: Dose response curves for the effect of neurotoxic agents on neurite outgrowth. hN2™ cells were cultured in the presence of cytotoxic compounds for 72h, stained for β-lll tubulin, imaged with the ImageeXpress and analyzed for neurite outgrowth.

I am always available for product related issues and questions. I cam be reached at direct phone: 612-801-1007 and pshuster@neuromics.com. Pete Shuster, CEO and Owner, Neuromics.

Save 100 USD on Our New Cardiomyocytes

Potent, Pure and Easy to Culture

I am pleased to announce the addition of Human Cardiomyocytes to our Stem and Cell Based Assay Solutions.

Image: Human Cardiomyocyte culture.

These cells are optimized to provide addition options for in-vitro testing of drug to drug candidate toxicities allowing researchers to rule out the ineffective and potentially toxic small molecules/compounds early in the process.

I want to make it easy for you to buy and try. If you are not delighted with your results, I will replace free of charge or refund your purchase. Please also note the select positive feedback on our other primary and stem cells:
We recently used the hMSCs derived from Umbilical Cord Blood. Their performance was nothing less than excellent. We were highly impressed with their morphology and their doubling rate. In addition the cells respond very well to accutase and maintain their performance after passaging. We highly recommend this cell line. Rodney Nash, Ph.D, Georgia State University.
Combined Hippocampus, Cortex, and Ventricular Neurons: "I got 10 million cells total after extraction from the tissue. At Day 4 they all developed long axons. Thank you so much for the replacement." Dr. Lidia Gardner, University of Tennessee HSC.
Neuromics always provides excellent products and the best customer service. I highly recommend this company's antibodies and neuronal cultures. Kirsten Raehal, Purdue Pharma

I am always available for product related issues and questions. I cam be reached at direct phone: 612-801-1007 and pshuster@neuromics.com. Pete Shuster, CEO and Owner, Neuromics.

hN2 Primary Human Neurons and Toxicity Assays

Neuromics' hN2 Cells Continue to Shine

I previously posted our Human hN2 Neurons being used for studying the mechanisms of  Nerve Agent VX: hN2 Human Neurons for Toxicity Screening. The versatility of these neurons enabled the researchers to perform a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Solid primary cell based assay results start with healthy and well behaving cells.

I am pleased to announce success in exposing the cells to a specific cytotoxicity inducing compound (small molecule): Mark RichardsChee Wee PhoonGwendoline Tze Wei GohEng Khuan SengXu Ming GuoCherine Mei Fong TanWoon-Khiong ChanJoel Mun Kin Lee. A New Class of Pluripotent Stem Cell Cytotoxic Small Molecules. Research Article | published 19 Mar 2014 | PLOS ONE 10.1371/journal.pone.0085039.

Image: Cytotoxic agent used in dose-response curve

Figure: Dose response curves for 3 specialized somatic cell lines (MRC-5, human primary neurons and human neonatal cardiomyocytes) treated with JC011.

This curve illustrates the sensitivity of human neurons to toxic agents. 

We plan on continuing to use these and our hNP1 Human Neural Progenitors in kinetic, "in vivo like" assays. These assays will give quantitative data on both growth and differentiation inducing agents as well as specifics on how the cells behave when exposed to toxic agents. I will be posting results here.

This data should be of interested to neuro-disease/disorders basic and drug discovery researchers. We plan on making the assays available to researchers. We currently also do small molecule testing and gene expression analysis studies a CRO offering. To learn more, I can be reached at pshuster@neuromics.com or 612-801-1007.

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.