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5 ARTICLES PUBLISHED IN JoVE

Medicine

Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

Anna U. Eriksson^*1, Christoffer Svensson^*1, Andreas Hörnblad¹, Abbas Cheddad¹, Elena Kostromina¹, Maria Eriksson¹, Nils Norlin¹, Antonello Pileggi², James Sharpe³, Fredrik Georgsson⁴, Tomas Alanentalo¹, Ulf Ahlgren¹

¹Umeå Centre for Molecular Medicine, Umeå University, ²Cell Transplant Center, Diabetes Research Institute, University of Miami,, ³EMBL-CRG Systems Biology Program, Centre for Genomic Regulation, Catalan Institute of Research and Advanced Studies, ⁴Dept. of Computing Science, Umeå University

We describe the adaptation of optical projection tomography (OPT)¹ to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic β-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

Chemistry

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

Samantha M. Desmarais¹, Felipe Cava², Miguel A. de Pedro³, Kerwyn Casey Huang^1,4

¹Department of Bioengineering, Stanford University, ²Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, ³Campus de Cantoblanco, Universidad Autonoma de Madrid, ⁴Department of Microbiology and Immunology, Stanford University School of Medicine

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

Immunology and Infection

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

Faezzah Baharom¹, Gregory Rankin², Saskia Scholz¹, Jamshid Pourazar², Clas Ahlm³, Anders Blomberg², Anna Smed-Sörensen¹

¹Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet, ²Division of Medicine, Department of Public Health and Clinical Medicine, Umeå University, ³Divison of Infectious Diseases, Department of Clinical Microbiology, Umeå University

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

Cancer Research

CAM-Delam Assay to Score Metastatic Properties by Quantifying Delamination and Invasion Capacity of Cancer Cells

Tami Green¹, Lina Šlekienė^1,2, Lena Gunhaga¹

¹Umeå Centre for Molecular Medicine, Umeå University, ²Department of Histology and Embryology, Medical Academy, Lithuanian University of Health Sciences

The CAM-Delam assay to evaluate the metastatic capacity of cancer cells is relatively fast, easy, and cheap. The method can be used for unraveling the molecular mechanisms regulating metastasis formation and for drug screening. An optimized assay for analyzing human tumor samples could be a clinical method for personalized cancer treatment.

Immunology and Infection

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

Anne Berthold^*1, Marie-Line Faucillion^*2, Ingela Nilsson², Maryna Golovchenko³, Vett Lloyd¹, Sven Bergström², Natalie Rudenko³

¹Department of Biology, Mt. Allison University, ²Department of Molecular Biology, Umeå University, ³Biology Centre Czech Academy of Sciences, Institute of Parasitology

In vitro culture is a direct detection method for the presence of living bacteria. This protocol describes methods for the culture of diverse Borrelia spirochetes, including those of the Borrelia burgdorferi sensu lato complex, relapsing fever Borrelia species, and Borrelia miyamotoi. These species are fastidious and slow growing but can be cultured.