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Authors: Dr. Steven Wilhelm, Samantha Coy
Group: VERVE Net, The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols
Summary: Ultracentrifugation in the WX Ultra Series can support only 30 mL samples at a time, and therefore do not significantly concentrate viruses. Tangential flow filtration enables viral concentration of large volumes that can then be purified using sucrose density gradients. Contact Dr. Steven Wilhelm ([email protected]) or Samantha Coy ([email protected]) for additional information regarding this protocol.

Proper citation: Dr. Steven Wilhelm, Samantha Coy 2017. Concentrating Viruses by Tangential Flow Filtration. protocols.io https://dx.doi.org/10.17504/protocols.io.hgrb3v6 Copy   

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Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. Diabetic Complication:

Proper citation: Eva Feldman 2019. Biochemical Measures of Neuropathy - Aconitase. protocols.io https://dx.doi.org/10.17504/protocols.io.3n4gmgw Copy   

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Authors: Lawrence M Berger, Giulia Ferrari, marion.leturcq , lidia.panico , Anne Solaz
Summary: Replication files for: COVID-19 Lockdowns and Demographically-Relevant Google Trends: A Cross-National Analysis.

Proper citation: Lawrence M Berger, Giulia Ferrari, marion.leturcq , lidia.panico , Anne Solaz 2021. replication_files_Berger-et-al_COVID_GOOGLE. protocols.io https://dx.doi.org/10.17504/protocols.io.br3xm8pn Copy   

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Authors: Faezah Mohd Salleh, Jazmin Ramos-Madrigal, Fernando Penaloza, Shanlin Liu, Mikkel-Holger S Sinding, Riddhi P Patel, Renata Martins, Dorina Lenz, Jorns Fickel, Christian Roos, Mohd Shahir Shamsir, Mohammad Shahfiz Azman, Burton K Lim, Stephen J Rossiter, Andreas Wilting, M Thomas P Gilbert
Group: GigaScience Press
Summary: This protocol provides an efficient DNA extraction and purification of historical museum hides, which potentially have been chemically tanned. 

Proper citation: Faezah Mohd Salleh, Jazmin Ramos-Madrigal, Fernando Penaloza, Shanlin Liu, Mikkel-Holger S Sinding, Riddhi P Patel, Renata Martins, Dorina Lenz, Jorns Fickel, Christian Roos, Mohd Shahir Shamsir, Mohammad Shahfiz Azman, Burton K Lim, Stephen J Rossiter, Andreas Wilting, M Thomas P Gilbert 2017. Extraction method C (FMS). protocols.io https://dx.doi.org/10.17504/protocols.io.inbcdan Copy   

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Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: This recepe is used in the following protocols:- Separation and purification of human PBMC from FRESH BLOOD- Separation and purification of human PBMC from BUFFY COAT- Magnetic bead-based CD4+ T cell isolation from PBMCs with Dynabeads: CD4 Positive Isolation Kit- Magnetic bead-based TREG-TEFF cell isolation from PBMC with Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit- Staining of human PBMC or ISOLATED SUBSETS with Cell Proliferation Dye-eFluor™ 670 (CPD-eFluor670) for cell proliferation evaluation by Flow Cytometry

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. SOLUTION- 02 - Phosphate Buffered Saline (PBS). protocols.io https://dx.doi.org/10.17504/protocols.io.biahkab6 Copy   

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Authors: Soon Kiat Lau, Jeyamkondan Subbiah

Proper citation: Soon Kiat Lau, Jeyamkondan Subbiah 2020. TDT Sandwich: An Open Source Dry Heat System for Characterizing the Thermal Resistance of Microorganisms. protocols.io https://dx.doi.org/10.17504/protocols.io.36agrae Copy   

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Authors: Chang-Ming Bai, Lu-Sheng Xin, Umberto Rosani, Biao Wu, Qing-Chen Wang, Xiao-Ke Duan, Zhi-Hong Liu, Chong-Ming Wang
Group: GigaScience Press
Summary: Background: The blood clam, Scapharca (Anadara) broughtonii, is an economically and ecologically important marine bivalve of the Family Arcidae. The efforts that have been made to study their population genetics, breeding, cultivation and stock enrichment were somewhat hindered by the lack of a reference genome. Here, we reported the complete genome sequence of S. broughtonii, a first reference genome of the Family Arcidae.Funding: A total of 75.79 Gb clean data was generated with the PacBio and Oxford Nanopore platforms, which represented approx. 86× coverage of the S. broughtonii genome. De novo assembly of these long reads resulted in an 884.5 Mb genome, with a contig N50 of 1.80 Mb and scaffold N50 of 45.00 Mb, respectively. Genome Hi-C scaffolding resulted in 19 chromosomes containing 99.35% of bases of the assembled genome. Genome annotation revealed that a considerable part of the genome (46.1%) is composed by repeated sequences, while 24,045 protein-coding genes were predicted and 84.7% of them were annotated.Conclusion: We report here the chromosomal-level assembly of the S. broughtonii genome based on long read sequencing and Hi-C scaffolding. The genomic data could be served as reference genome for the Arcidae Family and will provide a valuable resource for the scientific community and aquaculture sector.

Proper citation: Chang-Ming Bai, Lu-Sheng Xin, Umberto Rosani, Biao Wu, Qing-Chen Wang, Xiao-Ke Duan, Zhi-Hong Liu, Chong-Ming Wang 2019. Key protocols for chromosome-level genome assembly of the Scapharca (Anadara) broughtonii.. protocols.io https://dx.doi.org/10.17504/protocols.io.zimf4c6 Copy   

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Authors: Sam Li
Group: BioLegend
Summary: T regulatory cells (also known as Tregs or Regulatory T cells) are essential cells in the immune system that suppress immune responses of other cells, designed to limit excessive reactions and prevent autoimmunity. Tregs are characterized by the expression of CD4, CD25, and FOXP3, while lacking CD127. CD4+FOXP3+ regulatory T cells have been referred to as "naturally-occurring" regulatory T cells to distinguish them from "suppressor" T cell populations that are generated in vitro. While other variants of suppressive T cells do exist, such as CD8 suppressor cells, Th3 and Tr-1 cells, Tregs are classically defined as CD4+CD25+FOXP3+ cells. Here, we provide a protocol for immunofluorescent staining of FOXP3 in frozen sections, adapted from a protocol from Dr. Matthias Hardtke-Wolenski, Medizinische Hochschule Hannover. This protocol has been successfully used on mouse spleen, liver, and skin sections.

Proper citation: Sam Li 2019. Immunofluorescent Staining of Foxp3 in Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.97ch9iw Copy   

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Authors: Manuel Liebeke
Summary: TAHS was synthetized according to the protocol previously described with minor modifications. TAHS is used as derivatization reagent for amino acids. The purpose is to generate enough TAHS for on-tissue derivatisation to enhance signals during MALDI-MSI of amino acids from tissue sections.

Proper citation: Manuel Liebeke 2019. Synthesis of derivatization reagent TAHS (N,N,N-trimethylamonioanilyl N-hydroxysuccinimidyl carbamate iodide). protocols.io https://dx.doi.org/10.17504/protocols.io.j8ycrxw Copy   

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Authors: LI-COR Biosciences
Group: LI-COR Biosciences
Summary: This is a complete apoptosis assay example. It details the seeding, induction, and detection of the HeLa cellular response to Anisomycin treatment. Developed for: Aerius, Odyssey® Classic, Odyssey CLx, and Odyssey Sa Infrared Imaging Systems Please refer to your manual to confirm that this protocol is appropriate for the applications compatible with your Odyssey Imager model.

Proper citation: LI-COR Biosciences 2018. In-Cell Western™ Assay (HeLa Cellular Response to Anisomycin Treatment). protocols.io https://dx.doi.org/10.17504/protocols.io.gwibxce Copy   

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Authors: Trina Knotts
Group: Mouse Metabolic Phenotyping Centers
Summary: The work of Gordon and colleagues (i.e., Nature. 2006 Dec 21;444(7122):1027-31) has shown that obesity can result in marked shifts in the gut microbiome in mice and other models including humans. While the role of the microbiome remains to be fully elucidated, the gut microbiota can no longer be ignored as a potentially important factor when assessing metabolic phenotype. This service involves 16S gene variable region (V1-V3 or V3-V4 directed primers) sequencing by Titanium 454 (10,000 avg seq read depth) of feces, cecal, or other GI contents. Sequences can be processed through a bioinformatics pipeline (Qiime) to taxonomically classify them and to assess alpha and beta diversity of the community. In addition, Principal Components Analysis (PCA) or partial least squares- discriminant analysis (PLS-DA) can leverage variances in the relative microbial abundances to better understand how specific microbes contribute to separation by group. Correlational analyses can identify which variables of host metadata associate with specific microbes. The Core’s gut microbiome assay will employ this approach to uncover unique microbiota fingerprints in test mice. One caveat is that with fecal samples, patterns are only a surrogate for actual gut microbiota patterns, and may not exactly reflect the intestinal populations.

Proper citation: Trina Knotts 2019. UC Davis - Gut Microbiome Analysis (454-10K reads). protocols.io https://dx.doi.org/10.17504/protocols.io.ykjfuun Copy   

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Authors: Blue Lake, Kun Zhang
Group: Human Cell Atlas Method Development Community, KPMP, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Nuclei can be readily isolated from frozen tissues with a combination of chemical and physical treatments that can circumvent the non-uniform or incomplete dissociation of solid tissues into single cells. The isolation of nuclei can also circumvent RNA degradation or any introduction of technical artefacts (such as stress responses) that could be triggered during whole cell dissociation methods. Data generated from single-nucleus genomic assays permits discovery of molecular cell types that can be used to define the overall cellular makeup of a tissue or organ, and ultimately will inform upon adult human tissue atlases.

Proper citation: Blue Lake, Kun Zhang 2019. Isolation of single nuclei from solid tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.ufketkw Copy   

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Authors: Sean Seaver
Summary: Blue/White Screening of Bacterial Colonies X-Gal/IPTG Plates

Proper citation: Sean Seaver 2014. Blue/White Screening of Bacterial Colonies X-Gal/IPTG Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.cjauid Copy   

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Authors: Nam JH, Kim DH, Yoo JH, Hwang JH, Chang JD
Summary: Background Elderly patients undergoing hip fracture surgery (HFS) are at increased risk of postoperative venous thromboembolism (VTE). Therefore, combined postoperative mechanical and chemical thromboprophylaxis has been routinely performed after HFS in these patients. This retrospective case-control study was conducted to evaluate the additional effectiveness of preoperative mechanical thromboprophylaxis on the prevention of VTE following HFS in elderly patients.Methods Of 539 consecutive patients aged 70 years or older undergoing HFS, 404 (control group) did not receive preoperative mechanical thromboprophylaxis while 135 (study group) received mechanical thromboprophylaxis using an intermittent pneumatic compression device and graduated compression stockings from the time of admission until surgery. All patients received combined postoperative mechanical and chemical thromboprophylaxis following HFS in accordance with the same protocol. The incidence of symptomatic VTE confirmed by clinical symptoms and 3-dimensional CT angiography within one month of surgery was investigated in both groups.Results American Society of Anesthesiologists grade was higher (p=0.016) in the study group and more patients in this group had concomitant cardiovascular and neurologic diseases (p=0.005 and p=0.009, respectively). Meanwhile, more patients in the study group had received anticoagulant medication preinjury owing to comorbidities (39% vs 28%, p=0.025). The overall incidences of symptomatic deep vein thrombosis (DVT) and pulmonary embolism (PE) were 7.4% and 3.7% in the control group and 2.2% and 1.5% in the study group, respectively. On multiple logistic regression, symptomatic DVT significantly reduced in the study group (OR 0.28, p=0.042), meanwhile there was no significant difference in the incidence of symptomatic PE between the two groups (p=0.223).Conclusions Preoperative mechanical thromboprophylaxis may confer an additional benefit by preventing postoperative VTE without adding more risk of perioperative bleeding in elderly patients with hip fracture.

Proper citation: Nam JH, Kim DH, Yoo JH, Hwang JH, Chang JD 2017. Preoperative mechanical prophylaxis in elderly patients with hip fracture. protocols.io https://dx.doi.org/10.17504/protocols.io.jfscjne Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2019. MojoSort™ Human anti-PE Nanobeads Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7aghibw Copy   

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Authors: Benoit Besson, Florian Sonthonnax, Magalie Duchateau, Youcef Ben Khalifa, Florence Larrous, Hyeju Eun, Véronique Hourdel, Mariette Matondo, Julia Chamot-Rooke, Regis Grailhe, Hervé Bourhy
Summary: 1st purification: a-FLAG (A2220; Sigma-Aldrich)2nd purification: GFP TRAP_A (ChromoTek)

Proper citation: Benoit Besson, Florian Sonthonnax, Magalie Duchateau, Youcef Ben Khalifa, Florence Larrous, Hyeju Eun, Véronique Hourdel, Mariette Matondo, Julia Chamot-Rooke, Regis Grailhe, Hervé Bourhy 2017. Tandem affinity purification of a bait host protein in presence of a viral protein. protocols.io https://dx.doi.org/10.17504/protocols.io.jeqcjdw Copy   

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Authors: Sebastiaan Kuiper
Group: iGEM Wageningen 2019
Summary: To observe the potential of defense mechanisms of either native or synthetic systems in Escherichia coli (and more) when incubated with a bacteriophage stock.

Proper citation: Sebastiaan Kuiper 2019. Growth curve analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.77jhrkn Copy   

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Authors: Melissa Pontes Pereira, María Cristina Vidal Pessolani
Summary: Goal:Immunohistochemistry (or IHC) is a method that allows demonstrating the presence and location of proteins in tissue sections.Day 1:Thaw the slides about 15 minutes.Wash the slides 1 x 15 minutes in PBS with gentle agitation.Wipe away excess liquid around the section on the glass slide with tissue paper.Encircle the tissue section or draw lines on both sides of the section and let dry (10-15 seconds). The PAP PEN is designed to provide a water repellant barrier when a circle is drawn around a specimen such as tissue sections or cells. The barrier of the PAP PEN retains antisera within the defined area and ensures that only the amount of antibody needed for sufficient reaction is used.Block in 5% normal serum with 2% BSA in PBS plus 0.3% Triton X-100 (block solution) for 4 hours at room temperature.Drain slides for a few seconds (do not rinse)Wash the slides 1 x 15 minutes in PBS with gentle agitation.Wipe away excess liquid around the section on the glass slide with tissue paper.Apply primary antibody diluted in block solutionIncubate overnight at 4°C Day 2Drain slides for a few seconds (do not rinse)Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in block solution, and incubate for 2 hours at room temperature. This step should be done in the dark to avoid photobleaching.Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply DAPI 1:100 in PBS 1X for 5 min at room temperature.Rinse in running tap distilled waterCoverslip with mounting medium.Day 2Drain slides for a few seconds (do not rinse)Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in block solution, and incubate for 2 hours at room temperature. This step should be done in the dark to avoid photobleaching.Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply DAPI 1:100 in PBS 1X for 5 min at room temperature.Rinse in running tap distilled waterCoverslip with mounting medium.Attention!! All incubations should be carried out in a humidified chamber to avoid drying of the tissue.ControlsTo estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen (for example, BrdU) having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control. For whole serum antibodies, use normal serum from an unimmunized animal of the same species as the primary antibody. If an isotype control is not available, a negative antibody control is recommended. Simply replace the primary antibody with antibody diluent. A positive tissue control is strongly recommended to ensure that the antibody is performing as expected. Depending on the experiment, it may also be useful to include a negative tissue control: a tissue in which the protein of interest is not expected to be found.Referenceswww.abcam.com/technical

Proper citation: Melissa Pontes Pereira, María Cristina Vidal Pessolani 2018. Immunohistochemistry (or IHC) Protocol for frozen sections. protocols.io https://dx.doi.org/10.17504/protocols.io.pkmdku6 Copy   

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Authors: Mª José Gómez Torres, Javier Rodríguez santero, Javier Gil Flores

Proper citation: Mª José Gómez Torres, Javier Rodríguez santero, Javier Gil Flores 2019. CUESTIONARIO PARA PARTICIPANTES EN PROGRAMAS DE INTERVENCIÓN SOCIOLABORAL. protocols.io https://dx.doi.org/10.17504/protocols.io.uxdexi6 Copy   

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Authors: Francois-Yves bouget
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol  describes the selection and growth of stable transformants in semi solid agarose medium. Developped initially for Ostreococcus tauri, it also works for Ostreococcus lucimarinus and Bathycoccus.

Proper citation: Francois-Yves bouget 2019. Selection of stable transformants in Ostreococcus tauri, Ostreococcus lucimarinus and Bathycoccus prasinos. protocols.io https://dx.doi.org/10.17504/protocols.io.zj2f4qe Copy   

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