5. Cell Lysate Analysis

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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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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