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Authors: Maria Streltsova, Sofya Erokhina, Leonid Kanevskiy, Dean Lee, William Telford, Elena Kovalenko
Summary: Here we present the simple and effective method for obtaining clones of NK cells, sorted according to a given set of surface markers. The lifespan of clones grown by this method can reach 14 weeks and cloning efficiency is up to 50%, depending on the original NK cell subset. Obtained clones are functionally active; they are able to lyse target cells and produce IFNγ. The principle of method includes single cell sorting and further clonal expansion in the presence of IL-2 and gene-modified K562 feeder cells. Our method allows to expand the progeny of a single NK cell up to 10-20×106 NK cells suitable to study phenotype, proliferative and functional activity of a certain NK cell clone.The work was supported by Russian Science Foundation, grant #16-15-00309.
Proper citation: Maria Streltsova, Sofya Erokhina, Leonid Kanevskiy, Dean Lee, William Telford, Elena Kovalenko 2018. Obtaining of NK cell clones using IL-2 and gene-modified K562 cells expressing membrane-bound IL-21. protocols.io dx.doi.org/10.17504/protocols.io.v87e9zn Copy
Authors: Aleksandra Kozyczkowska
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Proper citation: Aleksandra Kozyczkowska 2018. Stable tranfection of unicellular relative of animals, Corallochytrium limacisporum, using Lonza Nucleofector. protocols.io dx.doi.org/10.17504/protocols.io.r5ud86w Copy
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: Stock vitamin solution for ESAW Media for Marine Phytoplankton
Proper citation: Ashley Humphrey 2016. Vitamin Solution for ESAW Media for Marine Phytoplankton. protocols.io dx.doi.org/10.17504/protocols.io.gdcbs2w Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to produce AAV of any serotype with high purity and high titer.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. Production of AAV Virus by Iodixanol Gradient Ultracentrifugation. protocols.io dx.doi.org/10.17504/protocols.io.bdvxi67n Copy
Authors: Yeseong Kim, Fahmi Shibli, Takafumi Kubota
Summary: The review question: What is the clinical effectiveness of proton pump inhibitor (PPI) therapy on sleepdisturbances in patients with gastroesophageal reflux disease (GERD)?The following databases should be use: CENTRAL; MEDLINE; EMBASE; WHO-ICTRP; ClinicalTrials.gov.Types of study to be included: Prospective, randomized controlled trials describing PPI therapy for patients with GERD and sleep disturbances will be assessed for eligibility. Crossover studies, clusterrandomized studies, will be excluded. Studies conducted by quasi-experimental methods will be excluded. Retrospective studies will be excluded. No exclusion will be made for observation periods.Main outcome(s): Change in sleep time and quality.Additional outcome(s): Change in pH impedance parameters (acid exposure, number of refluxes episodes); Change in reflux symptoms; All adverse events: Proportion of people who developed an adverse event according to the original author's definition.We will perform meta-analysis of the above outcomes using random effect models.Besides, we will assess heterogeniety and publication bias.
Proper citation: Yeseong Kim, Fahmi Shibli, Takafumi Kubota 2021. Efficacy of proton pump inhibitor (PPI) therapy in treating sleep disturbances in patients with gastroesopahgeal reflux disease (GERD): a systematic review and meta-analysis. protocols.io dx.doi.org/10.17504/protocols.io.bsrpnd5n Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: NEBExpress® Ni Resin is an affinity matrix for the isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. It is intended for use in gravity or pressure flow columns, and batch purifications. NEBExpress Ni Resin is comprised of a highly uniform and stable chemical-tolerant resin, pre-charged with nickel ions on the matrix surface. It is resistant to a wide range of chemicals, including NaOH, EDTA, DTT and β-Mercaptoethanol.Purification of ≥10 mg His-tagged protein per 1 ml of resinIntended for use in gravity or pressure flow columns, and batch purificationsHigh specific binding of His-tagged proteins yielding purities of >95%Strong nickel ion binding provides excellent resistance to EDTA and reducing agents. Compatible with commercially available detergent-based cell lysis reagentsIsolation and purification of His-tagged fusion proteins under native or denaturing conditions
Proper citation: New England Biolabs 2020. NEBExpress Ni Resin Gravity Flow Typical Protocol (NEB #S1428). protocols.io dx.doi.org/10.17504/protocols.io.bfd7ji9n Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to produce crude preps of AAV of any serotype.
Proper citation: Allen Institute for Brain Science 2019. Production of Crude AAV Virus Extract. protocols.io dx.doi.org/10.17504/protocols.io.8djhs4n Copy
Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: K-mer-based approaches to determining sequence similiarity can be faster than traditional use of BLAST. This application uses Jellyfish to index host FASTA files, then uses the mode value (default 2) of the number of matching k-mers (default k=20) from the input sequence to determine whether the sequence is similar enough to the host sequence to be rejected. The output of the app is:"screened" directory containing the sequences from each file that were found to be dissimlar to the host"rejected" directory containing the sequence from each file that were too similar to the host"jf" directory containing Jellyfish indexes of each "host" file (useful for later runs with other files to skip recreating)"kmer" directory containing k-mers of query sequences and ".loc" file showing the number of ".kmer" lines associated to each sequence Code is freely available at Github.
Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. K-mer-based host screening. protocols.io dx.doi.org/10.17504/protocols.io.ehjbb4n Copy
Authors: Chu-Yu Chin, Sun-Yuan Hsieh, Vincent S. Tseng
Summary: The early assessment of disease risk is an emerging topic in medical informatics. If diseases are detected at an early stage, prognosis can be improved and medical resources can be used more efficiently. A number of recent studies have considered risk factor analysis approaches, such as association rule mining, sequential rule mining, regression, and medical expert advice. In this study, for improving disease risk assessment, non-negative matrix factorization and support vector machine (SVM) were integrated to discover important and implicit risk factors.To make the method easy to follow, here we provide an experimental protocal. This experimental protocal comprises three main stages: data preprocessing, risk factor optimization, and early disease risk assessment. To discover the optimized risk factors, the NMF algorithm with parameter optimization was used for constructing the NMF-based matrix. In the assessment model learning and early disease risk assessment stages, the machine learning classifier SVM was used for disease modeling with the NMF-based matrix, yielding the final disease risk assessment, which serves as an excellent reference for physicians and patients.
Proper citation: Chu-Yu Chin, Sun-Yuan Hsieh, Vincent S. Tseng 2018. Effective early disease risk assessment with matrix factorization on a large-scale medical database. protocols.io dx.doi.org/10.17504/protocols.io.rv2d68e Copy
Authors: Priyanka Mishra, Amit Kumar, Akshitha Nagireddy, Ashutosh K. Shukla, Velusamy Sundaresan
Proper citation: Priyanka Mishra, Amit Kumar, Akshitha Nagireddy, Ashutosh K. Shukla, Velusamy Sundaresan 2017. Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia,. protocols.io dx.doi.org/10.17504/protocols.io.h4rb8v6 Copy
Authors: Erica C. Nakajima, Ronald Karwoski, Fabien Maldonado, Srinivasan Rajagopalan, Tucker F. Johnson, Michael P. Frankland
Summary: Computer-Aided Nodule Assessment and Risk Yield (CANARY) is a novel computed tomography (CT) tool developed at Mayo Clinic (Rochester, MN) that characterizes early lung adenocarcinoma by detecting nine distinct voxel classes, representing a spectrum of lepidic to invasive growth, within an adenocarcinoma. CANARY characterization has been shown to correlate with ADC histology and patient outcomes.This protocol provides basic instructions for segmentation of lung adenocarcinoma on CT imaging. CANARY has been validated in lung adenocarcinomas less than 3cm in diameter
Proper citation: Erica C. Nakajima, Ronald Karwoski, Fabien Maldonado, Srinivasan Rajagopalan, Tucker F. Johnson, Michael P. Frankland 2018. CANARY Segmentation of Lung Adenocarcinoma. protocols.io dx.doi.org/10.17504/protocols.io.mrcc52w Copy
Authors: Sharifah Albraiki
Group: Beck Lab
Proper citation: Sharifah Albraiki 2019. ActA purification protocol. protocols.io dx.doi.org/10.17504/protocols.io.nkadcse Copy
Authors: Daniel Uysal, Philipp Erben
Proper citation: Daniel Uysal, Philipp Erben 2021. In silico and RT-qPCR analysis of the 8q22.2 region in muscle invasive bladder cancer. protocols.io dx.doi.org/10.17504/protocols.io.bmewk3fe Copy
Authors: Peng Xu, Venice Servellita, Krzysztof Langer, Dan Weisgerber, Gordon Murtaugh, Adam R Abate, Charles Chiu
Group: Coronavirus Method Development Community, UCSF
Summary: We have developed a highly sensitive and specific LAMP(loop-mediated amplification)-based assay for thedetection of SARS-CoV-2 that can potentially be used for at-home and point-of-care (POC) testing. The assayincludes only three simple steps: (1) heat inactivate the sample mixed with lysis buffer and add LAMP reagentmix, (2) incubate the reaction on a heating device, and (3) read the result by inserting a lateral flow strip intothe tube. Performance is comparable to gold-standard RT-PCR testing, as the assay is based on nucleic aciddetection rather than antigen detection which is used by the majority of lateral flow strip-based assays. There isminimal pre-processing involved and no bulky instrumentation needed, and non-lab professionals can followthe instructions and understand the results easily. The assay targets the SARS-CoV-2 nucleoprotein (N) geneand is capable of detecting as low as 4.0 copies per reaction (0.5 copies/μL) of SARS-CoV-2 RNA,demonstrating no cross-reactivity with a panel of 20 other respiratory pathogens. Furthermore, the test can berun on multiple sample types, including nasopharyngeal and/or oropharyngeal swab and saliva samples.Overall, this assay provides a rapid (sample-to-answer turnaround time of ~40 min), accurate, and affordablesolution to the challenge of at-home diagnostics for SARS-CoV-2.
Proper citation: Peng Xu, Venice Servellita, Krzysztof Langer, Dan Weisgerber, Gordon Murtaugh, Adam R Abate, Charles Chiu 2021. A lateral flow-based at-home test for detection of SARS-CoV-2. protocols.io dx.doi.org/10.17504/protocols.io.bs8bnhsn Copy
Authors: Daniel Marchal
Summary: Adapted from https://www.protocols.io/view/Gibson-Assembly-Protocol-E5510-imss45
Proper citation: Daniel Marchal 2018. Gibson Assembly in V. natriegens. protocols.io dx.doi.org/10.17504/protocols.io.uvdew26 Copy
Authors: Chee Lim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:The tail-cuff blood pressure measurement is a non-invasive test and the principle is the same as that of the inflatable blood pressure cuff used in the standard clinic visit. The Visitech system uses an LED light source to detect the pulse signal wave. Since the measurement is sensitive to motion artifact, the procedure requires training sessions to acclimate the mouse to the process.
Proper citation: Chee Lim 2019. Vandy - Tail-Cuff Blood Pressure. protocols.io dx.doi.org/10.17504/protocols.io.yymfxu6 Copy
Authors: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda
Group: Coronavirus Method Development Community
Summary: This protocol is folked from "ARTIC amplicon sequencing protocol for MinION for nCoV-2019" by Josh Quick to adapt it to illumina sequencers. In this Version V3, the reverse-transcription step was changed to use of NEB's LunaScript RT SuperMix Kit instead of Thermo Fisher's SuperScript IV Reverse Transcriptase as adapting to the recent update of ARTIC Network's protocol (LoCost). Tyson et al., Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore bioRxiv2020.09.04.283077;doi:https://doi.org/10.1101/2020.09.04.283077 Because the PCR products are fragmented before ligated with adapters, this protocol is compatible to many sequencing kits with various read lengths (75PE, 150PE, etc.) in Illumina machines. While the library preparation uses QiaSeq FX by Qiagen and is basically straight forward (as par kit instruction but modified to 1/4 scale), some tweaks for much of simplicity and speed were added.Change histories (V2): The amount of cDNA input to multiplex PCR is increased (now almost same amount to the ARTIC Network's original protocol) (Step 10). This change gives better results for samples with extremely low RNA copy. Amount of adapter solution input was corrected (Step 19).Change histories (V3): RT step has been changed to use of LunaScript (Tyson et al., 2020).Corrected typos and wrong descriptions about amount of reagents.Added a link to library quantification protocol.Other minor changes.Change histories (V3): RT step has been changed to use of LunaScript (Tyson et al., 2020).Corrected typos and wrong descriptions about amount of reagents.Added a link to library quantification protocol.Other minor changes.
Proper citation: Kentaro Itokawa, Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda 2020. nCoV-2019 sequencing protocol for illumina. protocols.io dx.doi.org/10.17504/protocols.io.bnn7mdhn Copy
Authors: James Thornton
Group: Hurwitz Lab, MetaFunc Course
Summary: This protocol will introduce a workflow for quality control and pre-processing of metagenomic sequence reads using FastQC for visualization and FastX Toolkit for editing the fastq files.
Proper citation: James Thornton 2016. MG_HW3: Quality Control and Pre-processing. protocols.io dx.doi.org/10.17504/protocols.io.fuwbnxe Copy
Authors: Amy Ehrlich, Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: Gut tissue (by region- e.g ileum & colon) will be opened along the mesenteric border and mounted in Ussing chambers (Physiologic Instruments, San Diego, CA, USA), exposing 0.3 cm² of tissue surface area to 2.5ml of oxygenated Krebs-glucose (10mM) and Krebs-mannitol (10mM) at 37°C on the serosal and luminal sides, respectively. The paracellular pathway and transcellular pathway will be measured as the flux of FITC-Dextran 4000 (FD-4, Sigma –Aldrich) and horseradish peroxidase (HRP Type VI, Sigma Aldrich), respectively. FD-4 (400µg/ml) and HRP (200µg/ml) will be added to the mucosal chamber and samples will be collected from the serosal chamber every 30 min for 2 hours. Concentration of FD-4 is measured via fluorescence at excitation 485 nm, emission 538 nm. O-dianisidine substrate is used to detect HRP at absorbance 450 nm.
Proper citation: Amy Ehrlich, Trina Knotts 2019. UC Davis - Ex vivo assessment of barrier function-gut permeability. protocols.io dx.doi.org/10.17504/protocols.io.yipfudn Copy
Authors: Wen Aw
Group: Cage Studies
Proper citation: Wen Aw 2018. Triglyceride Quantification. protocols.io dx.doi.org/10.17504/protocols.io.rv9d696 Copy
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