Lysosome Targeting RedGreen-assay: Selective Autophagy Sensing Assay for Mammalian Cells

Lysosome Targeting RedGreen-assay: Selective Autophagy Sensing Assay for Mammalian Cells

The means of autophagy is a necessary mobile mechanism, required to take care of basic cell well being by the elimination of dysfunctional organelles, such because the ER, peroxisomes and mitochondria, in addition to protein aggregates, and micro organism. Autophagy is an especially dynamic course of, and instruments are continually being developed to check the varied steps of this course of. This protocol particulars a technique to check the top steps of autophagy-lysosomal fusion and the formation of the autolysosome. Many strategies have been used to check the varied steps of the autophagy course of.

Here we describe the RedGreen-assay (RG-assay), an immunofluorescence-based method used to visualise the concentrating on of substrates to the autolysosome in reside cells. This method takes benefit of the low lysosomal pH and over-expression of a tandem GFP-mCherry tagged protein focused to an organelle of curiosity. While within the impartial cytosol or autophagosome, each GFP and RFP will fluoresce. However, inside the autolysosome, the GFP sign is quenched as a result of low pH surroundings and the RFP emission sign will predominate. This method is quickly quantifiable and amenable to excessive throughput experiments. Additionally, by tagging the GFP-RFP tandem fluorescent protein with organelle particular concentrating on sequences, it may be used to measure a variety of substrates of autophagy.

Quantifying the ratio of alternatively spliced mRNA variants of genes with identified various splicing variants is extremely related for many purposes. Herein, we describe the validation of a quantitative PCR design for the simplified quantification of identified mRNA splice variants. The assay makes use of a single-common primer pair, twin probe design for the willpower of splicing variants in a single nicely configuration. We used murine XBP-1 splicing variants, XBP-1S and XBP-1U, to validate and show the efficiency traits of this method. Using artificial XBP-1S and XBP-1U cDNA in addition to cDNA synthesized from mouse beta-cell line MIN6, we established the efficiency parameters and dynamic vary of the assay. Reliable quantification of each variants at various focus gradients was proven.

Platelet Isolation and Activation Assays

Platelets regulate hemostasis and are the important thing determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets flow into in an inactive state, however develop into activated in response to wreck to the endothelium, which exposes thrombogenic materials resembling collagen to the blood circulate. Activation leads to plenty of responses, together with secretion of soluble bioactive molecules through the discharge of alpha and dense granules, activation of membrane adhesion receptors, launch of microparticles, and externalization of phosphatidylserine.

These processes facilitate agency adhesion to websites of damage and the recruitment and activation of different platelets and leukocytes, leading to aggregation and thrombus formation. Platelet activation drives the hemostatic response, and likewise contributes to pathogenic thrombus formation. Thus, quantification of platelet-associated responses is vital to many pathophysiologically related processes. Here we describe protocols for isolating, counting, and activating platelets, and for the fast quantification of cell floor proteins utilizing circulate cytometry.

Extracellular vesicles (EVs) are produced by all domains of life together with Bacteria, Archaea and Eukarya. EVs are important for mobile physiology and comprise assorted cargo: virulence components, cell wall transforming enzymes, extracellular matrix parts and even nucleic acids and metabolites. While varied protocols for isolating EVs have been established for mammalian cells, the sphere is actively growing instruments to check EVs in different organisms.

In this protocol we describe our strategies to carry out density gradient purification of EVs in bacterial cells, permitting for separation of EV subpopulations, adopted by safety assays for EV cargo characterization. Furthermore, we devised a protocol which includes a fluorescent conjugate of fatty acids into EVs, the primary to permit reside-cell EV monitoring to look at launch of EVs, together with throughout an infection of mammalian cells by pathogenic micro organism. These protocols are highly effective instruments for EV researchers as they allow the statement of EV launch and the research of the mechanisms of their formation and launch.

 Lysosome Targeting RedGreen-assay: Selective Autophagy Sensing Assay for Mammalian Cells

Ex vivo Drosophila Wing Imaginal Disc Culture and Furin Inhibitor Assay

Furin is an evolutionarily conserved proprotein convertase (PC) household enzyme with a broad vary of substrates which might be important for developmental, homeostatic, and illness pathways. Classical genetic approaches and in vitro biochemical or cell organic assays recognized that precursor types of most development issue household proteins are processed by Furin. To quantitatively assess the potential function of Furin in cleaving and modulating intercellular dispersion of a Drosophila signaling protein, we developed a easy assay by combining genetics, ex vivo organ tradition, pharmacological remedy, and imaging analyses.

Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay Kit (25 assays)

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Description: Assay Kit for detection of Capase 9 activity in the research laboratory

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Caspase-3 DEVD-R110 Fluorometric and Colorimetric Assay Kit (100 assays)

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Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay Kit (100 assays)

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Description: Minimum order quantity: 1 unit of 1KIT

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  • EUR 1302.00
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  • Shipped within 1 week.

Caspase 3 Colorimetric Assay Kit

55R-1270 25 assays
EUR 277
Description: Assay Kit for detection of Capase 3 activity in the research laboratory

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Caspase 8 Colorimetric Assay Kit

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Description: Assay Kit for detection of Capase 8 activity in the research laboratory

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Description: Assay Kit for detection of Capase 6 activity in the research laboratory

Caspase 2 Colorimetric Assay Kit

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Description: Assay Kit for detection of Capase 2 activity in the research laboratory

Caspase 5 Colorimetric Assay Kit

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EUR 303
Description: Assay Kit for detection of Capase 5 activity in the research laboratory

Caspase 10 Colorimetric Assay Kit

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Description: Assay Kit for detection of Capase 10 activity in the research laboratory

Caspase 4 Colorimetric Assay Kit

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EUR 312
Description: Assay Kit for detection of Capase 4 activity in the research laboratory

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Caspase-5 Colorimetric Assay Kit

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Caspase-10 Colorimetric Assay Kit

K2197-100 100 assays
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Caspase-10 Colorimetric Assay Kit

K2197-200 200 assays
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Caspase-10 Colorimetric Assay Kit

K2197-400 400 assays
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Caspase-4 Colorimetric Assay Kit

K2199-100 100 assays
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Caspase-4 Colorimetric Assay Kit

K2199-200 200 assays
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Caspase-4 Colorimetric Assay Kit

K2199-400 400 assays
EUR 1114

Caspase-8 Colorimetric Assay Kit

K2013-100 100 assays
EUR 474

Caspase-8 Colorimetric Assay Kit

K2013-200 200 assays
EUR 696

Caspase-8 Colorimetric Assay Kit

K2013-400 400 assays
EUR 1086

Caspase-6 Colorimetric Assay Kit

K2015-100 100 assays
EUR 487

Caspase-6 Colorimetric Assay Kit

K2015-200 200 assays
EUR 696

Caspase-6 Colorimetric Assay Kit

K2015-400 400 assays
EUR 1101

Caspase-2 Colorimetric Assay Kit

K2017-100 100 assays
EUR 529

Caspase-2 Colorimetric Assay Kit

K2017-200 200 assays
EUR 725

Caspase-2 Colorimetric Assay Kit

K2017-400 400 assays
EUR 1156

Caspase-8 Colorimetric Assay Kit

K113-100
EUR 479

Caspase-8 Colorimetric Assay Kit

K113-200
EUR 620

Caspase-8 Colorimetric Assay Kit

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Caspase-6 Colorimetric Assay Kit

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Caspase-6 Colorimetric Assay Kit

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Caspase-6 Colorimetric Assay Kit

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Caspase-2 Colorimetric Assay Kit

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Caspase-2 Colorimetric Assay Kit

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EUR 615

Caspase-2 Colorimetric Assay Kit

K117-400
EUR 958

Caspase-3 Colorimetric Assay Kit

K2008-100 100 assays
EUR 474

Caspase-3 Colorimetric Assay Kit

K2008-200 200 assays
EUR 696

Caspase-3 Colorimetric Assay Kit

K2008-400 400 assays
EUR 1086

Caspase-1 Colorimetric Assay Kit

K2011-200 200 assays
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Caspase-1 Colorimetric Assay Kit

K2011-400 400 assays
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Caspase-3 Colorimetric Assay Kit

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Caspase-3 Colorimetric Assay Kit

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Caspase-3 Colorimetric Assay Kit

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Caspase-5 Colorimetric Assay Kit

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Caspase-5 Colorimetric Assay Kit

K123-200
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Caspase-5 Colorimetric Assay Kit

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Caspase-10 Colorimetric Assay Kit

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Caspase-10 Colorimetric Assay Kit

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Caspase-10 Colorimetric Assay Kit

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EUR 958

Caspase-4 Colorimetric Assay Kit

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Caspase-4 Colorimetric Assay Kit

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EUR 615

Caspase-4 Colorimetric Assay Kit

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Caspase-1 Colorimetric Assay Kit

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EUR 479

Caspase-1 Colorimetric Assay Kit

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Caspase-1 Colorimetric Assay Kit

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EUR 958

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Caspase 9 Fluorometric Assay Kit

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Description: Assay Kit for detection of Capase 9 activity in the research laboratory

Caspase 9 Assay Kit, green

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Caspase-9 Fluorometric Assay Kit

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Caspase-9 Fluorometric Assay Kit

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Caspase-9 Fluorometric Assay Kit

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Caspase-9 Fluorometric Assay Kit

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Caspase-12 Fluorometric Assay Kit

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Caspase-6 Fluorometric Assay Kit

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Caspase-2 Fluorometric Assay Kit

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Caspase-3 Fluorometric Assay Kit

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Caspase-1 Fluorometric Assay Kit

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Caspase-3 Fluorometric Assay Kit

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Caspase-10 Fluorometric Assay Kit

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Caspase-4 Fluorometric Assay Kit

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Caspase-12 Fluorometric Assay Kit

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Red Active Caspase-9 Staining Kit

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total collagen assay kit 96-assays

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total protein assay kit 96-assays

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soluble collagen assay kit 5x 96-assays

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total protein assay kit 2x 96-assays

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Glutathione Colorimetric Assay Kit

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Methylglyoxal Assay Kit (Colorimetric)

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The protocol herein describes easy methods to ex vivo tradition Drosophila wing imaginal discs expressing a fluorescently tagged Drosophila Fibroblast Growth Factor (FGF, Branchless/Bnl) over an extended time period within the presence of Furin inhibitors and monitor the cleavage and intercellular dispersion of the truncated Bnl components utilizing microscopy. Although the assay described right here is for assessing the impact of Furin inhibition on Bnl cleavage within the Drosophila larval wing imaginal disc, the precept and methodology can simply be adopted for every other alerts, tissue techniques, or organisms. This technique and protocol present an assay for analyzing Furin exercise on a particular substrate by straight visualizing the spatiotemporal distribution of its truncated components in an ex vivo-cultured organ.