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Authors: Victor Rodriguez
Summary: This protocol is designed to be able to extract and analyze the concentration of chlorophyll within a sample on a molecular level. The procedures of this protocol require using copper II sulfate and hydrochloric acid as a means of trituration to determine the approximate level a chlorophyll within a given sample.

Proper citation: Victor Rodriguez 2019. Determining Chlorophyll Concentration using CuSO4 Magnesium-Copper Exchange Titration. protocols.io https://dx.doi.org/10.17504/protocols.io.7xrhpm6 Copy   

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Authors: DAVID DUNIGAN AND IRINA AGARKOVA
Group: VERVE Net
Summary: For use in CviJI Purification From IL-3A Virus Infected NC64A Chlorella.

Proper citation: DAVID DUNIGAN AND IRINA AGARKOVA 2016. CviJI Assay Conditions. protocols.io https://dx.doi.org/10.17504/protocols.io.esqbedw Copy   

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Authors: Ken Christensen
Summary: A collection of public protocols used in the Christensen Lab.

Proper citation: Ken Christensen 2020. Basic Molecular Biology. protocols.io https://dx.doi.org/10.17504/protocols.io.bc64izgw Copy   

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Authors: Ying Fan, Andrew J.M. Howden, Dario R. Alessi, Esther M. Sammler
Group: asap
Summary: We describe a fast and efficient immunomagnetic negative isolation method to purify peripheral blood neutrophils for LRRK2 kinase pathway analysis in humans .justify:after { content: ""; display:inline-block; width: 100%; } Gain of kinase function mutations in the Leucine rich repeat kinase 2 (LRRK2) are associated with causing Parkinson’s disease. LRRK2 phosphorylates a subgroup of Rab GTPases and their phosphorylation levels mirror LRRK2 kinase activation status. Here, we describe a facile and robust method for isolating peripheral blood neutrophils by immunomagnetic negative selection, subsequent treatment with and without the specific LRRK2 kinase inhibitor MLi-2 to ensure that any effect seen is LRRK2 kinase dependent and cell lysis. Neutrophil lysates can then be used to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of its endogenous Rab GTPase substrates either by quantitative immunoblotting or targeted mass-spectrometry. The benefit of using peripheral blood neutrophils is that they are an abundant and homogenous white blood cell population that express relatively high levels of LRRK2, Rab10 and other Rab GTPases. The downside is their high intrinsic elastase serine protease activity that necessitates using the neurotoxic, but efficient serine protease inhibitor Diisopropyl fluorophosphate (DIFP) when lysing the cells. Using this assay, we have demonstrated significantly elevated LRRK2 kinase pathway activity in human peripheral blood neutrophils of patients carrying specific mutations such as LRRK2 R1441G (doi:https://doi.org/10.1101/2021.01.28.21249614) and VPS35 D620N (PMID:29743203). This assay can also be used for pharmacodynamic and target engagement studies of small molecule LRRK2 kinase inhibitors. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Ying Fan, Andrew J.M. Howden, Dario R. Alessi, Esther M. Sammler 2021. Dundee neutrophil isolation protocol (from whole blood). protocols.io https://dx.doi.org/10.17504/protocols.io.bnhtmb6n Copy   

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Authors: goncalo.doria
Group: XPRIZE Rapid Covid Testing
Summary: Instruction of use for Doctor Vida pocket - COVID-19 assay

Proper citation: goncalo.doria 2020. Doctor Vida Pocket - COVID-19 assay - Instructions for Use. protocols.io https://dx.doi.org/10.17504/protocols.io.bkc8kszw Copy   

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Authors: Charlie Mize, Raj Reddy, Purav Badani
Group: Canary, XPRIZE Rapid Covid Testing

Proper citation: Charlie Mize, Raj Reddy, Purav Badani 2020. Canary VOC testing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkm4ku8w Copy   

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Authors: Kurt Steinwascher
Summary: Female larvae of the Aedes aegypti mosquito outcompete male larvae for food.  Male larvae pupate earlier and at smaller masses in vials where competition is most intense.  Competition at the higher densities causes the food/larva to appear lower than the equivalent mg/larva at lower densities for both sexes.  The total food per vial appears to change the nature of the exploitative competition among females, which affects the competition among males.  No interference competition was observed; females may dominate the competition by their larger size (increased filtering ability) and by retaining food particles within their guts to extract a larger fraction of their nutrients.  Pupal mass for both sexes is primarily affected by food/larva; density affects the pupal mass through competition and the total food/vial (the interaction between food and density) and this interaction is different for the two sexes.  Age at pupation is more affected by density than by food/larva and the interaction between food and density also differs across the two sexes for this variable.  The nature of competition between and within the sexes at various food levels and densities is described.  The implications of this complex response to food level and density among larvae on the ecology of this species is considered. 

Proper citation: Kurt Steinwascher 2018. Competition among Aedes aegypti larvae in microcosms. protocols.io https://dx.doi.org/10.17504/protocols.io.tcweixe Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for the rapid generation of 3’ transcriptomic-NGS-ready- single-cell-libraries from pools of cells.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number's 1U01MH114812-01 and 1U19MH114830-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2021. 10Xv3.1 Genomics Sample Processing. protocols.io https://dx.doi.org/10.17504/protocols.io.brt5m6q6 Copy   

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Authors: Niels Appelman
Group: iGEM Wageningen 2019
Summary: This protocol can be used to encase LAMP mastermix and primers in paraffin.

Proper citation: Niels Appelman 2019. Encasing LAMP mixture in paraffin. protocols.io https://dx.doi.org/10.17504/protocols.io.8jbhuin Copy   

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Authors: Sebastian Pott, Michael Wasney, Nadia Khan
Group: Single_Cell_Uchicago
Summary: What follows is the protocol for performing single-cell Nucleosome Occupancy and Methylome sequencing on single nuclei (scNOMe-seq). This plate-based method, which can be used to simultaneously assess nucleosome occupancy and DNA methylation in single nuclei, draws upon the approach laid out in Luo et al., 2018. Everything from the initial nuclei sorting step to the final library preparation that directly precedes sequencing is included in this protocol. This protocol is optimized for non-neuronal nuclei and can be applied to a range of different cell types (e.g., intestinal organoid, fibroblasts).This is the method we are using and it is working.Adapted from "Robust single-cell DNA methylome profiling with snmC-seq2," by C. Luo et al., 2018, Nature Communications 9(1), pp. 1-6. Copyright 2018 by the authors.

Proper citation: Sebastian Pott, Michael Wasney, Nadia Khan 2020. Multiplexed scNOMe-seq protocol based on isolated single nuclei. protocols.io https://dx.doi.org/10.17504/protocols.io.bgfvjtn6 Copy   

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Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Summary: For Transcriptomics During One-Step Growth Curves for Cellulophaga Phages protocol.

Proper citation: Matthew Sullivan 2016. RNA Samples Steps. protocols.io https://dx.doi.org/10.17504/protocols.io.dev3e5 Copy   

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Authors: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro
Group: Ribeiro Lab
Summary: This protocol is part of the manuscript: Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.

Proper citation: Zita Santos, Patrícia Francisco, Margarida Anjos, Célia Baltazar, Ana Paula Elias, Gabriela Tondolo Fioreze, Pavel M. Itskov, Matthew D. W. Piper, Carlos Ribeiro 2017. Rearing of Drosophila on Holidic Media (HM) for feeding behavior assays. protocols.io https://dx.doi.org/10.17504/protocols.io.hhcb32w Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
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. ELISA for quantification of IL-31 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkemktc6 Copy   

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Authors: Pringle Lab, Christian Renicke
Group: Aiptasia-Symbiodiniaceae Model System
Summary: This protocol is kit-free and can be used to isolate high quality genomic DNA of predominantly Symbiodinium from symbiotic Aiptasia which can be used as e.g. PCR template for genotyping. It is based on the method described in Coffroth et al., 1992.

Proper citation: Pringle Lab, Christian Renicke 2018. 2×CTAB Protocol for Isolation of predominantly Symbiodinium DNA from symbiotic anemones. protocols.io https://dx.doi.org/10.17504/protocols.io.kzqcx5w Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. ELISPOT Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.98yh9xw Copy   

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Authors: Andrew Crowley
Group: Ackerman Lab
Summary: Default scale - 1 L (makes approx. 2.5 sleeves of plates)

Proper citation: Andrew Crowley 2019. Agar Plate Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.4c3gsyn Copy   

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Authors: Ronald W.A.L. Limpens, Eric J. Snijder, Abraham J. Koster, Montserrat Bárcena
Group: Coronavirus Method Development Community
Summary: This protocol allowed the subcellular localisation of viral RNA synthesis in coronavirus-infected cells by EM autoradiography, though it is largely applicable to other types of EM autoradiography samples. Newly synthesized viral RNA is metabolically labelled by providing the infected cells with tritiated uridine while arresting cellular transcription. Subsequently, the samples are processed for EM, sectioned and incubated with a photographic emulsion. Radioactive disintegrations from the labelled viral RNA generate signal around the radioactive source that, after development, becomes apparent as electron-dense grains that can be detected by transmission electron microscopy. The advantages of this approach are its high sensitivity and its compatibility with high-contrast EM sample preparation protocols. The protocol presented here is an adaptation of different methods and tips to EM autoradiography discussed in detail by Williams, in Glauert, 1977, Practical Methods in Electron Microscopy, Volume 6, Chapter 4.

Proper citation: Ronald W.A.L. Limpens, Eric J. Snijder, Abraham J. Koster, Montserrat Bárcena 2020. Subcellular localisation of newly synthesized viral RNA in coronavirus infection by EM autoradiography. protocols.io https://dx.doi.org/10.17504/protocols.io.bfrtjm6n Copy   

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Authors: Simeng Li

Proper citation: Simeng Li 2018. Immunocytochemistry Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.thwej7e Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2015. Mixture for PCR Amplification (E7370). protocols.io https://dx.doi.org/10.17504/protocols.io.cg7tzm Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes bioanalyzer operation. The Agilent Bioanalyzer is used as a quality control step for PCR and IVT products. Peak size, shape, and sample concentration are recorded and used to evaluate samples on a pass/fail level.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2020. 2100 Bioanalyzer Operation for PCR, GTP and IVT Products. protocols.io https://dx.doi.org/10.17504/protocols.io.bfzjjp4n Copy   

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