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Authors: Fatma Gomaa, Zhuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb

Proper citation: Fatma Gomaa, Zhuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb 2018. Protocol for Lucifersae Plasmid Prep. protocols.io https://dx.doi.org/10.17504/protocols.io.niadcae Copy   

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Authors: Alice C. Poirier, John S. Waterhouse, Mrinalini Watsa, Gideon A. Erkenswick, Laís A. A. Moreira, Jia Tang, Jacob C. Dunn, Amanda D. Melin, Andrew C. Smith
Summary: Chemosignals are mediators of social interactions in mammals, providing con- and hetero-specifics with information on fixed (e.g. species, sex, group and individual identity) and variable (e.g. social, reproductive and health status) features of the signaler. Yet methodological difficulties of recording and quantifying odor signals, especially in field conditions, have hampered studies of natural systems. We present the first use of the Torion® portable gas chromatography-mass spectrometry (GC-MS) instrument for in situ chemical analysis of primate scents. We collected and analyzed swab samples from the scent-glands and skin from 13 groups (57 individuals) of two sympatric species of wild emperor tamarins, Saguinus imperator, and Weddell’s saddleback tamarins, Leontocebus weddelli (Callitrichidae). In total, 11 compounds of interest (i.e. probably derived from the animals) could be detected in the samples, with 31 of 215 samples containing at least one compound of interest. The composition of these 31 samples varied systematically with species, group, sex and breeding status. Moreover, we tentatively identified seven of the compounds of interest as methyl hexanoate, benzaldehyde, ethyl hexanoate, acetophenone, a branched C15 alkane, 4-methoxybenzaldehyde and hexadecan-1-ol. As the field of primate semiochemistry continues to grow, we believe that portable GC-MS instruments have the potential to help make progress in the study of primate chemosignaling in field conditions, despite limitations that we encountered. We further provide recommendations for future use of the Torion® portable GC-MS for in situ analyses.

Proper citation: Alice C. Poirier, John S. Waterhouse, Mrinalini Watsa, Gideon A. Erkenswick, Laís A. A. Moreira, Jia Tang, Jacob C. Dunn, Amanda D. Melin, Andrew C. Smith 2021. Operating of the Torion® T-9 portable gas chromatograph-mass spectrometer (PerkinElmer) for the analysis of animal scent samples . protocols.io https://dx.doi.org/10.17504/protocols.io.brpqm5mw Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare Tris Buffered Saline (TNT). 10X TNT is the stock solution used to prepare 1X TNT, which equilibrates tissue and maintains pH in physiological range.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. Tris Buffered Saline (TNT). protocols.io https://dx.doi.org/10.17504/protocols.io.bgncjvaw Copy   

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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 (LPS- induced CXC chemokine), MCP-1 (monocyte chemotactic protein-1; CCL-2), M-CSF (macrophage colony stimulating factor), MIG (monokine induced by gamma interferon; CXCL- 9), MIP-1α (macrophage inflammatory protein alpha; CCL-3), MIP-1 (macrophage inflammatory protein beta; CCL-4), and MIP-2/RANTES (macrophage inflammatory protein 2- alpha; 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 - Cytokines Panel I - multiplex. protocols.io https://dx.doi.org/10.17504/protocols.io.xvnfn5e Copy   

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Authors: Norfitriah Mohamed Sohaimi, Mohd Hair Bejo, Abdul Rahman Omar, Aini Ideris, Nurulfiza Mat Isa

Proper citation: Norfitriah Mohamed Sohaimi, Mohd Hair Bejo, Abdul Rahman Omar, Aini Ideris, Nurulfiza Mat Isa 2019. PCR and cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.7zfhp3n Copy   

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Authors: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol outlines our analysis of the potential antibiotic resistance genes found within the skin virome. We start by visualizing the relative abundances of the top 10 antibiotic resistance categories (according to the CARD). We then quantify the number of unique antibiotic resistance gene categories annotated in the samples at each skin site, and also look at the percent CARD annotated ORFs of the overall ORFs present in each sample at each skin site.

Proper citation: HANNIGAN GD, GRICE EA, ET AL. 2016. Script R14: CARD Antibiotic Resistance Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ejnbcme Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Rapid and irreversible heat inactivation eliminates unwanted activityImproved storage stability versus native enzymeFaster reaction setup (no supplemental additives like zinc required) and shorter incubation timeFlexible reaction conditions (active in any restriction enzyme buffer, no clean-up required)Less enzyme required (high specific activity), resulting in a lower cost per reactionNo need for multiple phosphatases (Quick CIP removes 5′- and 3′- phosphates from DNA, RNA and dNTPs )Active on unincorporated dNTPs in PCR products - improves DNA sequencing and SNP analysisRecombinant for purity, consistency and value

Proper citation: New England Biolabs 2020. Enzymatic PCR Cleanup Protocol (NEB #M0525). protocols.io https://dx.doi.org/10.17504/protocols.io.7r4hm8w Copy   

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Authors: Patrick Tso, Dana Lee
Group: Mouse Metabolic Phenotyping Centers
Summary: Quantitative determinations of non-esterified fatty acids in plasma/serum/lymph will be made using the NEFA-HR enzymatic colorimetric method assay.

Proper citation: Patrick Tso, Dana Lee 2019. U Cinn - NEFA Concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.xmnfk5e 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 than 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 CD14 Nanobeads Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7ahhib6 Copy   

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Authors: Sandra L. Ross, Marika Sherman, Patricia L. McElroy, Julie A. Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara Arvedson
Summary: Protocol for detecting bystander cytotoxicity in mixed cultures of target-negative and target-positive cells treated with BiTE® antibody constructs. Two methods and one modification are described.

Proper citation: Sandra L. Ross, Marika Sherman, Patricia L. McElroy, Julie A. Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara Arvedson 2017. BiTE® bystander T cell-dependent cellular cytotoxicity (TDCC) assay. protocols.io https://dx.doi.org/10.17504/protocols.io.hwfb7bn Copy   

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Authors: Sanjay Srivatsan, Junyue Cao

Proper citation: Sanjay Srivatsan, Junyue Cao 2021. Single cell RNA sequencing library preparation (2-level sci-RNA-seq). protocols.io https:// Copy   

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Authors: Mark Kelly, Timothy P. Fitzgibbons
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: This is a Mouse model of Transverse aortic constriction (TAC). A commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure. Initially leading to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Resulting in cardiac dilatation and heart failure.

Proper citation: Mark Kelly, Timothy P. Fitzgibbons 2019. U Mass - Transverse Aortic Constriction. protocols.io https://dx.doi.org/10.17504/protocols.io.56ig9ce Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: A previous protocol was contaminated with IL-17. This is an improved version of an ELISA for quantification of Interleukin 23. It activates the production of IL-17A, which is a pro-inflammatory cytokine, produced by a group of T helper cell known as T- helper17 lymphocytes in response to the said IL-23 [1,2].Reference1. Cho ML, Kang JW, Moon YM, Nam HJ, Jhun JY, Heo SB, Jin HT, Min SY, Ju JH, Park KS, Cho YG, Yoon CH, Park SH, Sung YC, Kim HY (May 2006). Journal of Immunology. 176 (9): 5652–61. 2. Nobee A, Justiz-Vaillant A, Akpaka PE, Poon-King P (2016) Levels of Interleukin 17 and 23 in Patients with Systemic Lupus Erythematosus (SLE) in Trinidad and Tobago. Immunochem Immunopathol 2: 115. doi: 10.4172/2469- 9756.1000115

Proper citation: Angel Justiz-Vaillant 2020. Improved protocol of ELISA for determining the serum concentration of IL-23 in humans.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjzakp2e Copy   

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Authors: John Stack, Gary Cline
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure for determining the glycogen content within liver and muscle tissue.

Proper citation: John Stack, Gary Cline 2019. Yale - Tissue Glycogen. protocols.io https://dx.doi.org/10.17504/protocols.io.y39fyr6 Copy   

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Authors: Noah Forsat, Andriko Palmowski, Yannick Palmowski, Maarten Boers, Frank Buttgereit
Summary: Importance Elderly patients are underrepresented in clinical trials. Objective To identify barriers and strategies concerning recruitment and retention of elderly patients in clinical trials. Data Source MEDLINE (via PubMed), Cochrane Central Register of Controlled Trials (via Cochrane Library), Embase (via Ovid)Eligibility Publications stemming from original research that report on barriers of or strategies for better recruitment or retention of elderly people. Outcomes Barriers and strategies concerning recruitment and retention of elderly people in clinical trials.Critical Appraisal This study is about identifying barriers and solutions concerning recruitment and retention of elderly patients in clinical trials. Typical endpoints requiring a risk of bias appraisal such as ones that concern efficacy or safety will not be assessed, consequently, there will be no risk of bias assessments. Furthermore, a very heterogeneous set of studies will be included for which there exists no single risk of bias-assessment tool.

Proper citation: Noah Forsat, Andriko Palmowski, Yannick Palmowski, Maarten Boers, Frank Buttgereit 2019. Identifying barriers and solutions concerning the recruitment and retention of elderly people in clinical research: Protocol for a Systematic Review. protocols.io https://dx.doi.org/10.17504/protocols.io.4f6gtre Copy   

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Authors: Drew Gitomer
Summary: This is an adaptation of Yanovitzky & Weber - because we were interested adapting Yanovitzky and Weber's tool for criminal justice reform. All of the policymaking process stuff was kept in, but we modified the list of policy actors, and policy focus, and Appendix C. We also did our coding in NVIvo so removed Appendix D in the oritinal protocol

Proper citation: Drew Gitomer 2019. Criminal Justice Policy Document Analysis . Version 2. protocols.io https://dx.doi.org/10.17504/protocols.io.zhsf36e 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™ Nanobeads Column Protocol - 2. protocols.io https://dx.doi.org/10.17504/protocols.io.69ehh3e Copy   

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Authors: Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Jeff Hammerbacher
Group: Hammer Lab
Summary: A plate-based assay to estimate the cytotoxic activity of pmel-1 T cells against target cancer cells. The CytoTox-ONE Homogeneous Membrane Integrity Assay from Promega (Madison, WI) is a lactate dehydrogenase (LDH) release-based assay. Dying cells have damaged cell membranes causing cytosolic components, such as LDH, to leak into the culture media. The CytoTox-ONE assay uses LDH in the culture media to convert resazurin to fluorescent resofurin. The resulting fluorescent signal is proportional to the number of non-viable cells in a sample. One advantage to using this assay for T cell-mediated cytotoxicity is that the cancer cells do not need to be removed from the wells to measure cell death, which is especially convenient with adherent cancer cell types that would require trypsinization. However, we have found that the assay produces noisy data and a set of control wells with T cells alone is required to subtract background, overall increasing the number of replicates that are necessary. We had consistent results when culturing the pmel-1 T cells with hgp100 peptide-pulsed MC38 colon cancer cells. But we had inconsistent results using pmel-1 T cells and their natural target, B16-F10 melanoma cells. This setup could be adapted for T cells other than pmel-1 and their target cells. The ratio of T cells to cancer cells and the duration of co-culture would have to be optimized.

Proper citation: Elinor Gottschalk, Bulent Arman Aksoy, Pinar Aksoy, Jeff Hammerbacher 2019. In vitro pmel-1 T cell-mediated cytotoxicity assay with CytoTox-ONE Homogenous Membrane Integrity Assay (Promega). protocols.io https://dx.doi.org/10.17504/protocols.io.6j4hcqw Copy   

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Authors: Andrew Adey, Casey Thornton
Summary: High-throughput single cell genomic assays resolve the heterogeneity of cell states in complex tissues, however, the spatial orientation within the network of interconnected cells is lost. As cell localization is a necessary dimension in understanding complex tissues and disease states, we present a tool for highly scalable spatially-resolved single cell profiling of chromatin state. We use high density multiregional sampling to perform single-cell combinatorial indexing on Microbiopsies Assigned to Positions for the Assay for Transposase Accessible Chromatin (sciMAP-ATAC) to produce single-cell data of equivalent quality to non-spatial single-cell ATAC-seq.

Proper citation: Andrew Adey, Casey Thornton 2021. sciMAP-ATAC. protocols.io https://dx.doi.org/10.17504/protocols.io.brhxm37n Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction. Uses the Calf Intestinal Alkaline Phosphatase (CIP - M0290)

Proper citation: New England Biolabs 2014. Dephosphorylation using CIP in Restriction Enzyme Reaction (M0290). protocols.io https://dx.doi.org/10.17504/protocols.io.cdhs35 Copy   

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