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Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:This experiment provides the quantification of multiple cytokines and chemokines using multiplexed-Luminex technology based on beads containing specific antibodies. Serum cytokine levels reflect chronic or acute inflammation, and circulating cytokines and chemokines are altered in obesity. Cytokines Panel I include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, KC (IL-8 homologue), IL-9, IL-10 (interleukin-10), IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, TNFα (tumor necrosis factor alpha), IFNγ (interferon gamma), IP-10 (interferon gamma-induced protein 10; CXCL-10), Eotaxin (CCL-11), G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocyte macrophage colony stimulating factor), LIF (leukemia inhibitory factor), LIX (LPSinduced CXC chemokine), MCP-1 (monocyte chemotactic protein-1; CCL-2), M-CSF (macrophage colony stimulating factor), MIG (monokine induced by gamma interferon; CXCL9), MIP-1α (macrophage inflammatory protein alpha; CCL-3), MIP-1β (macrophage inflammatory protein beta; CCL-4), and MIP-2/RANTES (macrophage inflammatory protein 2alpha; CXCL-2). A service can be requested for all or any combination of listed cytokines/chemokines for customized multiplexed Luminex assay.

Proper citation: Jason Kim 2019. U Mass - Hemoglobin A1c. protocols.io https://dx.doi.org/10.17504/protocols.io.xy3fpyn Copy   

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Authors: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores
Group: Coronavirus Method Development Community
Summary: This is Part 4 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of Vaccine Candidate against COVID-19 in adults > 18 years of age"This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.Please note that this is an evolving document, to be versioned and updated, based on community feedback and new data.

Proper citation: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores 2020. Study Population (Part 4 of Phase 3 study of Vaccine Candidate for COVID-19). protocols.io https://dx.doi.org/10.17504/protocols.io.bj5xkq7n Copy   

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Authors: Jian Gong
Group: Bosak Lab
Summary: This protocol describes the adaption of a silica colorimetric assay originally described in Strickland and Parsons, 1972 (p65-70), modified for use on a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA), using standard 96-well plates for rapid measurements of 1 mL, diluted water samples. This assay measures the concentration of molybdate-reactive silica (monomeric and short polymers of silicic acid) in solution.Samples for this assay should be filtered (0.2 µm syringe filter) and placed in 2 mL microcentrifuge tubes. Avoid sample freezing. Store samples in the dark whenever possible.

Proper citation: Jian Gong 2020. Dissolved silica colorimetric assay using a plate reader (96-well plate). protocols.io https://dx.doi.org/10.17504/protocols.io.bd7ni9me Copy   

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Authors: Ashley Humphrey
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Trace metals solution to be used in ESAW Media for Marine Phytoplankton 

Proper citation: Ashley Humphrey 2016. Trace Metal Solution for ESAW Media. protocols.io https://dx.doi.org/10.17504/protocols.io.gdabs2e Copy   

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Authors: Ajit N Shah

Proper citation: Ajit N Shah 2019. AccuBlue®Broad Range RNA Quantitation. protocols.io https://dx.doi.org/10.17504/protocols.io.87bhzin Copy   

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Authors: Yu Lin Tan, Oriol Pavón Arocas, Lucille Duquenoy, Tiago Branco
Summary: Here we describe a protocol to perform fluorescence in situ hybridization (FISH) in thin sections from frozen murine brain tissue. The protocol can be divided in several days or can be completed in two successive days: one day for extracting and slicing the brain and a second one for the FISH.

Proper citation: Yu Lin Tan, Oriol Pavón Arocas, Lucille Duquenoy, Tiago Branco 2021. Fluorescence In Situ Hybridization (FISH - RNAscope) in mouse brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.bq2imyce Copy   

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Authors: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Caecitellus sp. is a small phagotroph belonging to the biocosoecid order of the stramenopiles. Caecitellus is found across global oceans but it's role in marine ecosystems, as with many heterotrophic flagellates, is unclear. We isolated a strain of Caecitellus (small subunit rRNA gene was 99.53% identical to Caecitellus parvulus and Caecitellus paraparvulus) from a contaminated culture of Oxyrrhis marina and assessed its amenability to transformation. Fluorescein isothiocyanate conjugated to dextran was successfully introduced into Caecitellus sp. using a high voltage exponential decay pulse (1000 V, 10 µF, ∞ Ω) from a BioRad GenePulser Xcell (cuvette width 0.2 cm). Cell viability was confirmed by observing motility and fluorescence 24 h after electroporation (Figure 1). Lower voltage pulses did not result in FITC uptake. Estimates of transformation effifiency were difficult to obtain due to the small size of the cells.Unfortunately, the isolated strain of Caecitellus perished in an incubator malfunction. However, these results may be transferable to Caecitellus parvulus, a model for heterotrophic flagellates.

Proper citation: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling 2019. Electroporation of Caecitellus sp. with FITC-dextran. protocols.io https://dx.doi.org/10.17504/protocols.io.35kgq4w Copy   

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Authors: Lucas Armitage, Mark Wallet
Summary: This protocol is a modification of a previously published method by Wilgenburg et al (PLoS One, 2013) to obtain monocytes and Dendritic cells (DCs) from induced Pluripotent Stem Cells (iPSCs) lines. 

Proper citation: Lucas Armitage, Mark Wallet 2018. The production of monocyte-derived dendritic cells from human iPSCs. protocols.io https://dx.doi.org/10.17504/protocols.io.ng8dbzw Copy   

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Authors: Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)

Proper citation: Steven Wilhelm 2017. Screening Sequencing Datasets for Marker Genes in CLC Genomics. protocols.io https://dx.doi.org/10.17504/protocols.io.g5vby66 Copy   

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Authors: Angel Justiz-Vaillant
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. IL-31 is produced mainly by Th2 cells and dendritic cells. It is a proinflammatory cytokine and a chemotactic factor that direct polymorphonuclear cells, monocytes, and T cells to inflammatory lesions. IL-31 induces chemokines production and synthesis of IL-6, IL-16, and IL-32. [1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. Copy of ELISA for quantification of IL-30 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj33kqqn Copy   

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Authors: Serena Ding
Group: Behavioural Genomics
Summary: For imaging aggregation behaviour of 40 young adult C. elegans on agar using the Twinnie TwinCam system. Worms are synchronised by bleaching and refeeding for 72 hours, and then 40 young adult hermaphrodites are transferred by glass pipette onto a 35 mm regular NGM plate for imaging for 1 hour at 9 fps.

Proper citation: Serena Ding 2018. Fluorescence aggregation imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.vx9e7r6 Copy   

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Authors: petercb3
Group: Coronavirus Method Development Community
Summary: Indication: Qualitative detection of the nucleocapsid protein antigen from SARS-CoV-2 in nasopharyngeal aspirate specimens directly or after the aspirate have been added to viral transport media from individuals who are suspected of COVID-19 by their healthcare provider. Emergency use of this test is limited to authorized laboratories and other authorized testing locations.

Proper citation: petercb3 2020. ClearLab Flu+COVID19 Antigen Assay IFU. protocols.io https://dx.doi.org/10.17504/protocols.io.bj8fkrtn Copy   

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Authors: Integrated Islet Distribution Program
Group: Integrated Islet Distribution Program
Summary: This Standard Operating Procedure is adapted from the work of the 'National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities'' following the SOP cited in the document 'Purified Human Pancreatic Islet - Viability Estimation of Islet Using Fluorescent Dyes (FDA/PI): Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium'This SOP defines the procedure for assessment of viability of human isolated islet preparations, which include endocrine and exocrine tissue, for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sponsored research in the Integrated Islet Distribution Program (IIDP). This protocol is written to assist the participating islet isolation centers and investigators who are part of this program.Fluorescein Diacetate/ Propidium Iodide (FDA)/(PI) Viability Assay is a rapid fluorometric method to test the integrity of the plasma membrane simultaneously using inclusion and exclusion dyes; the assay differentiates between viable and nonviable cells and is, consequently, used for determination of viability of islet preparations.

Proper citation: Integrated Islet Distribution Program 2020. Viability Estimation of Islets for Distribution Using Inclusion and Exclusion Fluorescent Dyes (FDA/PI). protocols.io https://dx.doi.org/10.17504/protocols.io.bhdmj246 Copy   

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Authors: Matthew Sullivan Lab
Group: Earth Microbiome Project

Proper citation: Matthew Sullivan Lab 2019. Quanti-iT™ Pico Green dsDNA Assay (Invitrogen P7589). protocols.io https://dx.doi.org/10.17504/protocols.io.c5zy75 Copy   

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Authors: Mariana Rius, Kashyapa Bandaralage
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Sonication procedure implemented to reduce labyrinthulomycete cell aggregation from culture for subsequent growth analyses. 

Proper citation: Mariana Rius, Kashyapa Bandaralage 2017. Dispersing Labyrinthulomycete Cell Aggregates by Sonication. protocols.io https://dx.doi.org/10.17504/protocols.io.hg2b3ye Copy   

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Authors: Susanna Sabin, James Fellows Yates
Group: WarinnerGroup, MPI-SHH Archaeogenetics
Summary: This protocol describes how to sample dental calculus from skeletal remains for biomolecular analysis. This protocol is particularly recommended for sampling calculus from teeth attached to a jaw bone, skull, or skeleton. The primary use-case is for DNA and proteomic analysis.

Proper citation: Susanna Sabin, James Fellows Yates 2020. Dental Calculus Field-Sampling Protocol (Sabin version). protocols.io https://dx.doi.org/10.17504/protocols.io.bqecmtaw Copy   

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Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for thymus dissociation (10-week old CD-1 female). 

Proper citation: Andrew Potter 2018. Adult mouse thymus dissociation (on ice). protocols.io https://dx.doi.org/10.17504/protocols.io.q7edzje Copy   

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Authors: Carolyn Elya, Ciera Martinez
Summary: Paraffin embedding and sectioning is a classic technique used in a variety of biological disciplines. Though the fruit fly Drosophila melanogaster are one of the most commonly-employed model organisms, the immense Drosophila literature is lacking in current protocols for sectioning whole, adult flies. Here, we adopted a plant histology protocol to embed entire adult flies for thin sectioning and present our protocol for performing FISH on the subsequent sections. In the interest of reproducibility, we have tried to include details that are normally omitted from published protocols.

Proper citation: Carolyn Elya, Ciera Martinez 2017. Paraffin embedding, microtomy and fluorescence in situ hybridization (FISH) of whole adult Drosophila. protocols.io https://dx.doi.org/10.17504/protocols.io.k5ecy3e Copy   

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Authors: Daubian Santos, Guilherme Cunha Ribeiro, Aline Diniz Cabral, Márcia Aparecida Sperança
Summary: Preparation of insect specimens for morphological studies classically employs chaotropic salts which can cause disruption of important structures. Also, extraction of nucleic acids for genetic studies leads to destruction of the specimen. Thus, in this paper is proposed a new technique, based on the use of proteinase K, which allows DNA extraction while keeping intact the entire morphology of insect specimens. The presented technique can contribute to taxonomic and systematic studies on different groups of arthropods. Also, with the global emergency and reemergence of diseases transmitted by arthropod vectors, genetic and morphological investigations performed in an individual-scale would unveil important aspects of environment and host-pathogen interactions.

Proper citation: Daubian Santos, Guilherme Cunha Ribeiro, Aline Diniz Cabral, Márcia Aparecida Sperança 2018. A non-destructive enzymatic method to extract DNA from a single arthropod specimen. protocols.io https://dx.doi.org/10.17504/protocols.io.kkqcuvw Copy   

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Authors: Marthe Howard
Summary: Wholemount Immunolabeling-GutThis protocol is used for immunostaining large blocks of tissue. The application that it was developed for is large pieces of mouse gut tissue. Tissues shold be well washed prior to fixation.

Proper citation: Marthe Howard 2019. Wholemount Immunolabeling for GUT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.wr6fd9e Copy   

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