Kidney-Chip

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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Fluorescence Imaging

Introduction

All fluorescent or confocal imaging of cells can be done directly in the Organ-Chip. You do not need to disassemble the chip, or isolate the membrane in order to image cells. The membrane is located 0.8 mm from the bottom of the chip and is visually accessible by most objectives on most microscopes, although the use of a long-working distance objective lens is recommended for optimal results. The chip is made of an optically transparent material that will not cause any significant distortion of your signal or image; it does not auto fluoresce.


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Fixation and Immunofluorescence (IF) Staining

Introduction

Goal: Fixing and IF staining of cells in Emulate Organ-Chips.

Note: This is the method we have developed for fixation and immunofluorescence staining for our OrganChips. We realize, however, that users may have their own fixation and staining processes that have been developed for specific cell types, antibodies, or antigens. If users would prefer to use other fixatives, permeabilizing solutions, or blocking buffers, they may do so while following the process outlined below. This protocol has been optimized for fixation and staining of the Emulate Liver-Chip for our specific staining protocols.


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