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GEN Protocols

Analytical Testing

Analytical Testing

Protocol

AAV Aggregate Quantitation and Identification with the Aura System

Adeno-associated viruses (AAVs) have made the promise of gene therapy a reality by enabling safe, targeted delivery of curative genetic payloads. The formation of aggregated viral vector is an important critical quality attribute to measure because they lead to adverse immunogenic events and are an ... More


Protocol

Detect Specific Protein Aggregates in Biologic Formulations with the Aura Immunoassay

Biologic co-formulations target multiple pharmacologically active sites to increase therapy efficacy. However, either biologic can be a source of protein aggregation that can negatively influence the product’s safety, efficacy, and therapy. Aura systems are the only particle analyzer that can pinpoi... More


Protocol

Evaluate Cell Therapy Product Purity with Aura CL

The CAR-T development workflow is a multi-step process that includes transfection of T cells with a viral vector to express a chimeric antigen and activation/expansion of the CAR-T using a molecule like a Dynabead conjugated to CD3/CD28. Each of these steps is a potential source of contamination tha... More


Protocol

USP Particle Count Standards with Aura and Particle Vue 3.1

All membrane microscopy methods must generate accurate and reproducible counts for bead counting standards. We present a validated methodology for accurately counting United States Pharmacopeia (USP) microsphere count standards on Aura systems by seamlessly calculating the cumulative area of all the... More


Protocol

USP Compendial Particle Measurements with the Aura System

The Aura platform provides a rapid and robust USP 788 compendial method for subvisible particle analysis, a critical quality attribute. It can handle a wide range of sample types and can analyze anywhere from 5 µL to 10 mL of sample, enabling continuity of method from developability assessment to lo... More


Protocol

Removal of rare undifferentiated induced pluripotent stem cells (iPSCs) by multi-step negative sorting

In the field of regenerative medicine involving ES or iPS cells, differentiated cells are often cultured to tissues and implanted to patients. However, there is an unneglectable problem that some remaining undifferentiated cells in the implanted tissue may cause tumors. Therefore, a complete removal... More


Protocol

Cell culture CO2 incubator decontamination process using hydrogen peroxide vapor for highly regulated and general cell culture protocols

The value of the laboratory cell culture incubator used in highly regulated research and clinical protocols is directly related to the proportion of incubator uptime vs. downtime in applications where frequent interior chamber decontamination is required or desired. The need for interior decontamina... More


Protocol

Performing a manual run on your Xuri™ Cell Expansion System W25

The purpose of this protocol is to provide instructions on how to perform a manual run on the Xuri™ Cell Expansion System W25 using UNICORN™ software.This protocol applies to anyone performing a manual run on the Xuri™ Cell Expansion System W25.... More


Protocol

Radiolabelling Nucleic Acids with [γ-32P] ATP for Analytical Assays

Nucleic acids are readily modified with tags that enable detection, purification, or tracking their interactions with other molecules. One of the standard labels used to generate nucleic acid probes includes radioactive phosphates because they allow for sensitive and rapid detection. This protocol d... More


Protocol

Preparing a Brain-Mimetic 3D Matrix for Culturing Human Pluripotent Stem Cells

This is a protocol for culturing human pluripotent stem cells (hPSCs) using a 3D matrix. It provides instructions for preparing a brain-mimetic 3D scaffold that can support neuronal growth and for culturing hPSCs. This injectable scaffold can be potentially employed in the field of regenerative medi... More


Protocol

Prevalence of SARS-CoV-2 Antibodies in Children and Adults with Type 1 Diabetes

Objective: As diabetes is a risk factor for severe symptoms, hospitalization, and death with COVID-19 disease, we aimed to assess the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in children and adults with and without type 1 diabetes in Colorado during 2020.... More


Protocol

Sustainable Use of a Real-Time Continuous Glucose Monitoring System from 2018 to 2020

We aimed to describe patterns of continuous glucose monitoring (CGM) system use and glycemic outcomes from 2018 to 2020 in a large real-world cohort by analyzing anonymized data from US-based CGM users who transitioned from the G5 to the G6 System (Dexcom) in 2018. The main end points were persisten... More


Protocol

Correlates of Continuous Glucose Monitoring Use Trajectories in Children and Adolescents with Type 1 Diabetes

The goal of this study was to characterize trajectories of continuous glucose monitoring (CGM) use in youth 5–12 weeks after starting CGM and examine what factors differentiate between the trajectory groups. Parent–youth dyads completed assessments before starting CGM. Days of CGM use between weeks ... More


Protocol

Continuous Glucose Monitoring Time-in-Range and HbA1c Targets in Pregnant Women with Type 1 Diabetes

The CONCEPTT trial compared real-time Continuous Glucose Monitoring (RT-CGM) to capillary glucose monitoring in pregnant women with type 1 diabetes. We analyzed CGM and glycated hemoglobin (HbA1c) measures in first (n = 221), second (n = 197), and third (n = 172) trimesters, aiming to examine target... More


Protocol

Insulin Metrics: Need for Development of Consensus Standards for Reporting of Insulin Dosing Data

The advent of connected insulin pens will generate an avalanche of digital insulin data, especially in the context of prandial- and multiple daily injection-insulin regimens. There is a need for the diabetes community to develop standards for such data, analogous to what has been achieved using the ... More


Protocol

Predictive Low-Glucose Suspend Necessitates Less Carbohydrate Supplementation to Rescue Hypoglycemia: Need to Revisit Current Hypoglycemia Treatment Guidelines

Current guidelines recommend 15–20 g of carbohydrate (CHO) for treatment of mild to moderate hypoglycemia. However, these guidelines do not account for reduced insulin during suspensions with predictive low-glucose suspend (PLGS). We assessed insulin suspensions, hypoglycemic events, and CHO treatme... More


Protocol

Development and Validation of an Anion Exchange High-Performance Liquid Chromatography Method for Analysis of Empty Capsids and Capsids Encapsidating Genetic Material in a Purified Preparation of Recombinant Adeno-Associated Virus Serotype 5

The development of various manufacturing platforms and analytical technologies has substantially contributed to successfully translating the recombinant adeno-associated viral vector from the laboratory to the clinic. The active deployment of these analytical technologies for process and product cha... More


Protocol

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography

We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing A... More


Protocol

Optimized Protocol for Accurate Titration of Adeno-Associated Virus Vectors

Adeno-associated virus (AAV) is currently the most popular gene delivery vector for in vivo gene therapy. However, variability in titration methods between different laboratories affects the reproducibility of experiments and evaluation of safety and efficacy in clinical trials. We describe an optim... More


Protocol

Cartilage Assessment Requires a Surface Characterization Protocol: Roughness, Friction, and Function

The surface of articular cartilage is integral to smooth, low-friction joint articulation. However, the majority of cartilage literature rarely includes measurements of surface characteristics and function. This may, in part, be due to a shortage of or unfamiliarity with fast, nondestructive, and, p... More


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Primer

Assessing Protein Purity: Many Tests, Many Assays

Proteins provide structure to cells, catalyse enzymatic reactions, transport cargo, replicate DNA, metabolise nutrients among an array of other functions. Composed of amino acids and generally featuring a three-dimensional structure, proteins form key therapeutic and diagnostic tools as antibodies o... More


Primer

Two Critical Steps to Success in Multicolor Flow Cytometry

Flow cytometry is a powerful tool to analyze and characterize cells based on their protein expression, most often those expressed on the cell surface. Multicolor flow cytometry uses multiple fluorochromes as primary or secondary antibody conjugates to label cells. The capacity for modern flow cytome... More


Primer

Considerations when Selecting a Matrix for MALDI

One of the most widely utilized techniques in mass spectrometry imaging (MSI) is label-free MALDI (Matrix-Assisted Laser Desorption Ionization), which can be coupled to a variety of mass analyzers to create brilliant molecular maps. Application targets include lipids, proteins, peptides, drugs, and ... More


Primer

Is Your Flow Cytometer Reliable?

Flow cytometry provides a useful method by which scientists can measure and analyse different properties of individual cells, including size, internal complexity (granularity) and the presence of cell surface and intracellular markers using fluorescence. Flow can help identify individual cell subpop... More


Primer

Unwanted Fungi: Detecting Microbial Cell Culture Contamination

Coming into the lab and spotting an obvious fungal growth, or the tell-tale signs of cloudy media, can be a stressful experience for researchers. When carrying out cell culture in the lab, contamination from microbial agents such as bacteria and fungus can occur and have disastrous effects on ongoin... More


Primer

Benefits of Using an Electronic Laboratory Notebook

Electronic laboratory notebooks (ELNs) were developed to keep track of experiments and data so that scientists could review their work in a single place and easily reproduce experiments. Many ELNs started out as electronic versions of paper lab notebooks. Now, the most advanced ELNs offer an extensi... More


Primer

Quick Antibiotic Susceptibility Test for High-Threat Pathogens in Whole Blood Samples

By spreading so rapidly and wreaking so much havoc, COVID-19 has revealed the susceptibility of global societies to both natural pathogens and intentionally released bioterror agents. The latter include bacteria that have been engineered for enhanced antibiotic resistance.1 Once pathogenic bacteria ... More


Primer

Bio-Electrospraying and Cell Electrospinning: A Sea of Applications

Electrospraying and electrospinning have been used for a wide range of applications ranging from coatings, propulsion, and pesticide sprays to scaffolding a wide range of advanced materials for applications in filtration and tissue engineering. Although electrospun scaffolds have been explored as ti... More


Primer

Techniques to Detect Unusual Nucleic Acid Structures

Guanine quadruplexes (G-quadruplexes or G4s) are unusual nucleic acid structures with distinct biological and chemical functions. They have occupied significant attention in the nucleic acid world since they were first identified in 1962 as the basis for the aggregation of 5’-guanosine monophosphate... More


Primer

Cloning Mice From Freeze Dried Somatic Cells

As species continue to face massive extinction, preserving genetic material through judicious biobanking to enable cloning is key to promoting the survival of species and maintaining biodiversity.Embryos and germ cells are commonly stored at ultra-low temperatures in liquid nitrogen. But storing rep... More


Mixer

Cell Therapy Development: Tackling Analytic Challenges

Cell therapy products are an exciting new class of medicinal products that offer opportunities to treat and cure diseases that cannot be treated using conventional therapeutic options. The development and manufacturing of cell therapy products pose several challenges compared to other biologics. Yet... More


Video

GEN Protocols Expert Exchanges: COVID-19 Immunodiagnostic Test Identifies Antibody Source

Need another COVID-19 booster?A new immunodiagnostic test called PictArray helps you decide. The test tells you exactly where your antibodies to the virus are coming from - whether your antibodies are from vaccines or SARS-CoV-2 infections, or both. The test detects SARS-CoV-2 antigens--both spike p... More