4. Effluent Analysis

Standard Curve Calculator

Introduction

The measurements made using many analytical instruments, like plate readers, require the conversion from a raw signal to a concentration or other known readout. For example, a ubiquitous Organ-Chip readout is the concentration of a given substance (either biological or pharmaceutical), in the Chip effluent. Typical assays on a plate reader, however, yield a raw signal, like optical density or fluorescence intensity. The conversion from signal intensity to the readout of interest is performed using a standard curve.

The standard curve process requires: first, the preparation of solutions of known concentrations; second, the quantification of the signal intensity of those solutions on an analytical instrument like a plate reader; and third, the establishment of a relationship between the measured signal and known concentrations through some curve fitting method. This relationship can then be used to convert the measured signal intensity from experimental samples to the particular readout, like concentration.

Like other standard curve calculators, the calculator within this excel can be used to establish the relationship between the measured signal from an analytical instrument to known concentrations. After entering the measured signal and the corresponding known concentrations in the "Standard Curve" tab, a curve is fit to the data using a log-log linear regression, which minimizes the percent error between the data and the fitted curve. This ensures a "best-fit" for both relatively low and high concentrations. The equation corresponding to this curve is displayed on a plot of the data/best-fit curve and additionally saved in the calculator's memory to convert the signal from experimental samples of unknown concentrations.

To analyze the results from an entire plate, simply copy and paste plate reader results/signal into the tab marked "Measurement"; the calculator will take the equation found in the "Standard Curve" tab and apply it to the signal in order to automatically calculate and display the corresponding readout. While this readout will usually be concentration, this calculator applies broadly to any readout and any signal. The results can be copied and pasted for further processing and analysis (e.g. using an output like "effluent concentrations" of a tracer molecule to then calculate apparent permeability).


← Back to Protocols

Barrier Function Analysis

Introduction

The maintenance or disruption of tissue barriers is an essential part of the pathophysiology of many diseases. The ability to quantitatively characterize tissue barrier is critical in the evaluation of barrier integrity and function.

This protocol is to be used to assess the permeability of an Organ-Chip's endothelial-epithelial barrier. Apparent permeability (Papp) of tracer molecules is determined by dosing the inlet of one channel, collecting the effluent of both channels, and calculating the amount of compound that crossed through the membrane over time. See full method below and associated Papp Calculator (EC004) for data analysis.


← Back to Protocols

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.


← Back to Protocols

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.


← Back to Protocols

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.


← Back to Protocols