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Thermo Fisher Scientific

8 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Genotyping of Plant and Animal Samples without Prior DNA Purification
Pak Y. Chum 1, Josh D. Haimes 1, Chas P. André 1, Pia K. Kuusisto 1, Melissa L. Kelley 1
1Thermo Scientific Molecular Biology Products, Thermo Fisher Scientific

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

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Developmental Biology

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells
Scott J. Callahan 1,2, Yvonne Mica 3, Lorenz Studer 1
1The Center for Stem Cell Biology, Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 2Cancer Biology and Genetics Program, Gerstner Sloan-Kettering Graduate School, Sloan-Kettering Institute for Cancer Research, 3Thermo Fisher Scientific

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

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Developmental Biology

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming
Rene H. Quintanilla Jr. 1, Joanna Asprer 1, Kyle Sylakowski 1, Uma Lakshmipathy 1
1Life Sciences Solutions, Thermo Fisher Scientific

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

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JoVE Core

Power Input Measurements in Stirred Bioreactors at Laboratory Scale
Stephan C. Kaiser 1, Sören Werner 2, Valentin Jossen 2, Katharina Blaschczok 2, Dieter Eibl 2
1Finesse, Thermo Fisher Scientific, 2Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, School of Life Sciences and Facility Management

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

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Immunology and Infection

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR
Suman K. Pradhan 1, Jerry Guzman 1, Carl Dargitz 1, Stephanie Switalski 2, Mark Landon 1, Sven Olek 3, Bjoern Samans 3, Ulrich Hoffmueler 3, Uma Lakshmipathy 1
1Cell Biology, Life Sciences Solutions, Thermo Fisher Scientific, 2California Polytechnic State University (Cal Poly), 3Epiontis GmbH, Precision for Medicine

Here we describe a robust method of determining immune cell identity and purity through epigenetic signatures detected using quantitative PCR (qPCR). DNA demethylation at a specific locus serves as a unique identifier for a particular cell type and allows for identification of CD8+, regulatory, or Th17 T cells.

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Engineering

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve
Yifei Liu 1, Yong-Ung Lee 2, Tai Yi 2, Ken Wu 3, Cedric Bouchet-Marquis 3, Han Chan 3, Christopher K. Breuer 2, David W. McComb 1
1Center for Electron Microscopy and Analysis, The Department of Materials Science and Engineering, Ohio State University, 2Center for Regenerative Medicine, Nationwide Children’s Hospital, 3Thermo Fisher Scientific

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

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Biochemistry

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope
Adrian Koh *1, Sagar Khavnekar *2, Wen Yang 1, Dimple Karia 1, Dennis Cats 1, Rob van der Ploeg 1, Fanis Grollios 1, Oliver Raschdorf 1, Abhay Kotecha 1, Daniel Němeček 3
1Materials and Structural Analysis Division, Thermo Fisher Scientific, 2Max Planck Institute of Biochemistry, Molecular Structural Biology, 3Materials and Structural Analysis Division, Thermo Fisher Scientific

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

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Biochemistry

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow
Alan Tin Lun Lam 1, Premkumar Jayaraman 2, Abigail Becker 3, Ryan Lim 2, Kim Leng Teo 1, Jacqueline Ng 2, Steve Oh 1
1Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A STAR), 2Thermo Fisher Scientific, 3Thermo Fisher Scientific

Here, we present a protocol to harvest adherent cells from multi-layered flasks in a closed semi-automated manner using a counterflow centrifugation system. This protocol can be applied for harvesting both adherent and suspension cells from other cell expansion platforms with few modifications to the existing steps.

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