Alkaline Phosphatase (ALP) Assay

Introduction

Alkaline phosphatases (ALPs) are a family of cell surface glycoproteins with ALP isoenzymes expressed in kidney as well as in a variety of other organs such as liver, intestine, and bone. In the clinic, elevated levels of serum ALP are associated with disease or injury in serval organs. It can be applied as a marker of kidney injury and disease. This protocol uses the Proximal Tubule Kidney-Chip as a reference point, and these assay conditions could change with a different Organ-Chip.


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Total ROS and RNS Assay

Introduction

Accumulation of free radicals, coupled with an increase in oxidative stress, has been implicated in the pathogenesis of several disease states and in the mechanism of action for toxicity of many compounds. This protocol can be used to measure these toxicity endpoints. The OxiSelect™ In Vitro ROS / RNS Assay Kit allows for the measurement of the total amount of free radicals in cell effluent by using a specific ROS / RNS fluorogenic probe. The fluorescence intensity is proportional to the total ROS / RNS levels within the sample.

This protocol uses the Proximal Tubule Kidney-Chip as a reference point. These methods and assay conditions could change with different Organ-Chips.


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Creatinine Quantification Assay

Introduction

Creatinine, a non-protein nitrogenous (NPN) waste product, is produced from the breakdown of creatine and phosphocreatine. Creatinine levels can be used as an indicator of renal function.

To measure creatinine secretion in the Proximal Tubule Kidney-Chip, the vascular channel (bottom channel) is perfused with medium containing 1mg / dL of human creatinine, while the secretion of creatinine in the epithelial channel (top channel) is measured from chip effluent that collects in outlet reservoirs of the Pod™ portable module.

This protocol uses the Proximal Tubule Kidney-Chip as a reference point. These methods and assay conditions could change with different Organ-Chips.


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Live Staining of CLF Uptake into Bile Canaliculi

Introduction

Corning® Cholyl-lysyl-fluorescein (CLF) staining is used to visualize the structure and function of bile canaliculi in polarized hepatocytes. CLF is a substrate for the canalicular bile salt export pump (BSEP), thus it can be used to visualize BSEP-mediated canalicular efflux, as well as to label the bile canaliculi structures.


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Human Cleaved Caspase-3 (Asp 175) Quantification

Introduction

The Cleaved Caspase-3 (Asp175) SimpleStep ELISA® kit is designed for the quantitative measurement of Active Caspase-3 (Asp175) protein in human cells. Caspase-3 is a cytoplasmic cysteine protease involved in the activation cascade of caspases responsible for cell apoptosis.

The SimpleStep ELISA uses an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocaptures the sample analyte in solution. This three-part complex is then immobilized via immunoaffinity of an anti-tag antibody coated on the well.

This endpoint can be applied to measure apoptosis in Organ-Chips as part of toxicity testing or other types of studies.


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Gsh-Glo™ Glutathione Assay

Introduction

The GSH-Glo™ Assay is a luminescent-based assay for the detection and quantification of glutathione (GSH), an antioxidant that can prevent damage to cellular components caused by reactive oxygen species such as free radicals, peroxides, and lipid peroxides. GSH is involved in the detoxification of both xenobiotic and endogenous compounds. A change in GSH levels can be used as an indicator of toxicity.


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Glutathione S-Transferase (αGST) Assay

Introduction

TECO® αGST is an ELISA for the quantitative determination of alpha Glutathione S-Transferase (αGST). GSTs are enzymes that catalyze the conjugation of glutathione to electrophilic centers on a wide variety of substrates in order to make the compounds more water-soluble. The role of GSTs is to detoxify endogenous compounds such as those that enable the breakdown of xenobiotics. High intracellular concentrations of GSTs coupled and released into serum are indicators of hepatocyte injury and toxicity and can be used as biomarkers. They are also used as indicators of renal injury and nephrotoxicity.


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Keratin 18 (K18) Assay

Introduction

Keratin 18 (K18) is an intracellular protein expressed at high levels by many types of epithelial cells. During cell death, the cellular content of soluble K18 will be released into the extracellular compartment. The M65 EpiDeath® ELISA measures soluble keratin 18 (K18) (cytokeratin 18 [CK18]) released from dying cells and can be used in the research of overall cell death (due to apoptosis and necrosis) of epithelial cells.


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