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Authors: Cristina Hernandez Rollan, Kristoffer Bach Falkenberg, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm
Summary: This protocol describes the expression and extraction of heterologously cytoplasmically expressed proteins in the bacterium E.coli, for T7 based expression systems. Specifically, it describes how to produce "hard-to-produce", as for example, LPMOs.It is based on a modified version of:"IPTG Induction and Extraction of Proteins from Bacteria" by Swathi Arur and Sudhir Nayak, Schedl Lab. Washington University Genetics, St. Louis. Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126.The extraction of cytoplasmatic proteins is followed by a chemical lysis protocol, alternatively, sonication can also be employed.
Proper citation: Cristina Hernandez Rollan, Kristoffer Bach Falkenberg, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm 2020. Protein expression of hard-to-produce proteins in the cytoplasm of Escherichia coli. protocols.io dx.doi.org/10.17504/protocols.io.bfbwjipe Copy
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: Qualitative and Quantitative Assessment of Islets Using Dithizone (DTZ) – Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium'This SOP defines the assay method for quantitative and qualitative determination in the identification 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.Dithizone (diphenyl thiocarbazone; DTZ) is an organic chemical that chelates the zinc in the insulin granules present in the beta cells of the pancreatic islets. The islet cells are stained red while the acinar cells remain unstained.DTZ staining is used as a lot release and as an in-process assay: (i)Lot release testing: DTZ staining is used to identify islets and to determine the quantity and quality of the final islet product. Islet quantity is expressed as the number of islet equivalents (IEQ), which is calculated based on the number and diameter of the islets present in the preparation, mathematically corrected for islet volume. (ii)In-process testing: DTZ staining is used to identify islets and to assess the effectiveness of the digestion, isolation and purification processes. The quality of the preparations is expressed as percent islet purity, and percent trapped islets. Islet quantity (IEQ) is also assessed.
Proper citation: Integrated Islet Distribution Program 2019. Qualitative & Quantitative Assessment of Human Islets for Distribution Using Dithizone (DTZ). protocols.io dx.doi.org/10.17504/protocols.io.5yvg7w6 Copy
Authors: Thomas Tamras, Tim Baker, Helle Mölsted Alvesson, Martin Gerdin Wärnberg
Summary: IntroductionPatients who are critically ill, those who are in need of critical care, can be found all over hospitals. Some, but not all, receive care in ICUs (intensive care units). Medical specialties usually define themselves by organ system, disease process or procedure, however, critical care has struggled to define its identity. (1) Critical care medicine, when used synonymous to intensive care medicine, emerged as a result of multiple historical events. Knowledge regarding trauma, shock and infectious medicine grew as the World War II and the conflicts in Korea and Vietnam occurred. The poliomyelitis epidemic during the 1950s led to the development of invasive mechanical ventilation for patients with respiratory failure. Technological developments enabled critical care medicine to emerge and develop. (2) Today, critical care medicine is considered to be resource-intensive and the National Health Services in England spends approximately £1 billion annually. (3)However, as initially alluded to, critical care can also be understood as something broader than critical care medicine or intensive care medicine. Critical care can be given to critically ill patients admitted to the ICU or wherever else there are patients who are critically ill, meanwhile critical care medicine or intensive care medicine is usually only received in the ICU.A retrospective study by Halpern et al published in 2004 investigated the role, use and costs of critical care in the Unites States between 1985 and 2000. Critical care medicine (CCM) beds in the United States increased by 26.2% between 1985 and 2000, during the same period non-CCM beds decreased by 30.9%. (4) There is an absence of a consistent definition of critical care. Some countries focus on the capability to support failing organ systems, or on the ratio between nurses and patients, where other countries focus on the need for close monitoring. There is a need for a universal consensus regarding the definition regarding of what constitutes critical care. A universal definition would greatly benefit the increasing need for critical care research and clinical discussion. (5) AimThe aim of this study is to operationally define critical care. The review will attempt to answer the question: What are the main elements of existing definitions of critical care and can these be homogenized to form a common definition?DesignThis scoping review will be conducted to examine how the literature defines critical care. The scoping review complemented by a thematic content analysis. A scoping review methodology is an approach to synthesizing available evidence and is particularly suited for questions not answerable by a systematic review because the scope is too broad, or when not much has been published on the topic. Both these conditions apply in this case. The review will be conducted using the Arksey and O’Malley framework and hence first relevant studies will be identified, second studies will be selected for inclusion, third data will be charted and finally, the data will be summarized. (6, 7) This protocol is structured following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) checklist (8).References
1. Adhikari N, Fowler R, Bhagwanjee S, Rubenfeld G. Critical care and the global burden of critical illness in adults. The Lancet. 2010;376(9749):1339-1346.
2. Kelley M. Critical Care Medicine — A New Specialty?. New England Journal of Medicine. 1988;318(24):1613-1617.
3. Amiruddin N, Prescott G, Coventry D, Jansen J. Evaluating service development in critical care: The impact of establishing a medical high dependency unit on intensive care unit workload, case mix, and mortality. Journal of the Intensive Care Society. 2018;19(3):226-235.
4. Halpern N, Pastores S, Greenstein R. Critical care medicine in the United States 1985–2000: An analysis of bed numbers, use, and costs*. Critical Care Medicine. 2004;32(6):1254-1259.
5. Wunsch H, Angus D, Harrison D, Collange O, Fowler R, Hoste E et al. Variation in critical care services across North America and Western Europe*. Critical Care Medicine. 2008;36(10):2787-e8.
6. Arksey H, O'Malley L. Scoping studies: towards a methodological framework. International Journal of Social Research Methodology. 2005;8(1):19-32.
7. Levac D, Colquhoun H, O'Brien K. Scoping studies: advancing the methodology. Implementation Science. 2010;5(1).
8. Tricco A, Lillie E, Zarin W, O'Brien K, Colquhoun H, Levac D et al. PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Annals of Internal Medicine. 2018;.
Proper citation: Thomas Tamras, Tim Baker, Helle Mölsted Alvesson, Martin Gerdin Wärnberg 2018. Defining critical care – a scoping review and thematic content analysis: protocol for a scoping review. protocols.io dx.doi.org/10.17504/protocols.io.twcepaw Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Proper citation: New England Biolabs 2015. PNGase F Non-Denaturing Mixture. protocols.io dx.doi.org/10.17504/protocols.io.cqjvum Copy
Authors: Vivian Liu
Summary: This protocol is for whole protein extraction from tissues like kidney, liver ,musscle etc. Histone proteins which are tightly twisted on genomic DNA can also be extracted with this protocol.
Proper citation: Vivian Liu 2015. Whole protein extraction from tissues. protocols.io dx.doi.org/10.17504/protocols.io.dkx4xm Copy
Authors: LI-COR Biosciences
Group: LI-COR Biosciences
Summary: This protocol describes a cell viability assay that uses near-infrared fluorescent detection. Sapphire700 Stain is used to determine cell viability by assessing cell membrane integrity, and the assay is imaged with the Odyssey CLx Imaging System.
Proper citation: LI-COR Biosciences 2018. Cell Viability Assay on Saponin-treated A431 Cells. protocols.io dx.doi.org/10.17504/protocols.io.gstbwen Copy
Authors: David Booth
Summary: In this lab, you will use immunofluroescence stainging to visualize the wondrous cellular transformations that occur throughout sea urchin development. The goals of this module are1. To learn about marine organisms at Marine Resources and some of their biology2. Understand the steps of immunofluorescence and its relative advantages3. Visualize stained embryos and understained the basic tradeoffs of some types of imaging techniques
Proper citation: David Booth 2019. Immunofluorescence Staining of Sea Urchin Embryos. protocols.io dx.doi.org/10.17504/protocols.io.388grzw Copy
Authors: Paulo A. Garcia, Zhifei Ge, Laura Kelley, Steven Holcomb, Cullen R. Buie
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Synthetic biology holds great potential for addressing pressing challenges for mankind and our planet. One technical challenge in tapping into the full potential of synthetic biology is the low efficiency and low throughput of genetic transformation for many types of cells. In this paper, we discuss a novel microfluidic system for improving bacterial electrotransformation efficiency and throughput. Our microfluidic system is comprised of non-uniform constrictions in microchannels to facilitate high electric fields with relatively small applied voltages to induce electroporation. Additionally, the microfluidic device has regions of low electric field to assist in electrophoretic transport of nucleic acids into the cells. The device features hydro- dynamically controlled electric fields that allow cells to experience a time dependent electric field that is otherwise difficult to achieve using standard electronics. Results suggest that transformation efficiency can be increased by ∼4×, while throughput can increase by 100–1000× compared to traditional electroporation cuvettes. This work will enable high-throughput and high efficiency genetic transformation of microbes, fa- cilitating accelerated development of genetically engineered organisms.
Proper citation: Paulo A. Garcia, Zhifei Ge, Laura Kelley, Steven Holcomb, Cullen R. Buie 2017. High Efficiency Microfluidic Electrotransformation. protocols.io dx.doi.org/10.17504/protocols.io.hapb2dn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol provides step by step instructions for performing single-cell sorting by using BD FACSAria II machine and BD FACSDiva software in preparation for single-cell sorting.
Proper citation: Allen Institute for Brain Science 2019. BD FACS Aria II Start-up and Calibration. protocols.io dx.doi.org/10.17504/protocols.io.bamdic26 Copy
Authors: Sakari Joenvaara, Mayank Saraswat, Pentti Kuusela, Shruti Saraswat, Rahul Agarwal, Johanna Kaartinen, Asko Jarvinen, Risto Renkonen
Summary: This protocol describes the complete protocol for performing N-Glycoproteomics from plasma samples. Albumin removal is performed prior to protein digestion with trypsin. Tryptic peptide mixture is used for enriching N-glycopeptides by lectin-affinity chromatography using a lectin-mix. Enriched N-glycopeptides are cleaned up by reverse phase chromatogrraphy and analyzed by LC-MS and LC-MS/MS. resulting MS/MS spectra is deconvouted and saved as peak lits file which can be searched with GlycopeptideId software to identify the peptide sequence, glycan composition and proposed glycan struccture.
Proper citation: Sakari Joenvaara, Mayank Saraswat, Pentti Kuusela, Shruti Saraswat, Rahul Agarwal, Johanna Kaartinen, Asko Jarvinen, Risto Renkonen 2018. Targeted N-glycoproteomics of human plasma samples.. protocols.io dx.doi.org/10.17504/protocols.io.ncadase Copy
Authors: Darach Miller
Summary: The aim of this protocol is to describe how to do a very reproducible upshift of a nitrogen-limited yeast culture to nitrogen non-limited conditions. In order to do this with enough cells to make inefficient analysis (ie RATEseq) feasible, we start in the chemostat for high-cell counts.
Proper citation: Darach Miller 2016. Nitrogen-upshift, chemostat to batch mode. protocols.io dx.doi.org/10.17504/protocols.io.ffhbjj6 Copy
Authors: Seth Currlin, Marda Jorgensen, Jerelyn Nick
Group: Optical Clearing of Tissue, Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: Tissue preparation for CLARITY includes fixation in 4% PFA, infusion with monomers in Hydrogel Solution, and clearing of lipids in Electrophoretic Tissue Clearing Solution.
Proper citation: Seth Currlin, Marda Jorgensen, Jerelyn Nick 2019. Tissue Preparation for CLARITY. protocols.io dx.doi.org/10.17504/protocols.io.8jihuke Copy
Authors: Kelley Knizner, Christopher Simmons
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: This method details sectioning of the FFPE HuBMAP specimens.Protocol adopted from Leigh Propper, Marda Jorgensen (12/13/2019). HuBMAP Tissue Sectioning for FFPE Specimens. https://dx.doi.org/10.17504/protocols.io.bahdib26
Proper citation: Kelley Knizner, Christopher Simmons 2020. HuBMAP | Sectioning of FFPE Specimens. protocols.io dx.doi.org/10.17504/protocols.io.bqqbmvsn Copy
Authors: Sandra Belalcázar-Rey, Valeria Sánchez, Juan C Ochoa-Tabares, Samuel Altamirano, Abraham Soto-Gómez, Ruben Suárez-Velasco, Filiberto García-Félix., Leopoldo Baiza-Durán, Oscar Olvera-Montaño, Patricia Muñoz-Villegas.
Summary: The present study was designed to evaluate the efficacy of a sodium hyaluronate/chondroitin sulfate, preservative-free, ophthalmic solution (SH/CS-PF) in normalizing the parameters of the ocular surface, alleviating symptoms and in reducing the squamous metaplasia using the conjunctival impression cytology (CIC) in patients with mild-moderate dry eye disease (DED).
Proper citation: Sandra Belalcázar-Rey, Valeria Sánchez, Juan C Ochoa-Tabares, Samuel Altamirano, Abraham Soto-Gómez, Ruben Suárez-Velasco, Filiberto García-Félix., Leopoldo Baiza-Durán, Oscar Olvera-Montaño, Patricia Muñoz-Villegas. 2019. Efficacy and safety of the PRO-087 Ophthalmic solution versus systane(R) Ultra and Systane(R) Ultra Preservative-free on the tear film dysfunction syndrome from mild to moderate.. protocols.io dx.doi.org/10.17504/protocols.io.3wvgpe6 Copy
Authors: Iciar Arteagoitia
Summary: Data sources: Pubmed, Cochrane, Web of Science, Science Direct, Scopus, and Ovid MD. Searched until 30th April 2017. (chlorhexidine) AND (bacteremia OR bacteraemia) AND (extraction OR removal) were used as key words in a free-text search. Meeting Abstracts published were searched. References of each article were reviewed. We only included randomized controlled clinical trials. There were no restrictions regarding language or date of publication. The outcome measure was set on the incidence of the bacteremia measure within the first ten minutes post-extraction. Two reviewers independently undertook the risk of bias assessment and data extraction. A fixed-effect inverse variance weighted meta-analysis was conducted.
Proper citation: Iciar Arteagoitia 2018. Does chlorhexidine reduce bacteremia following tooth extraction?. Systematic Review and Meta-analysis. protocols.io dx.doi.org/10.17504/protocols.io.m65c9g6 Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Preparation of feeder-free iPSCs culture. protocols.io dx.doi.org/10.17504/protocols.io.x83fryn Copy
Authors: Bosak Lab Protocols
Group: Bosak Lab
Proper citation: Bosak Lab Protocols 2020. BG11 hypersaline agar plates. protocols.io dx.doi.org/10.17504/protocols.io.bkchkst6 Copy
Authors: Bioline
Group: Bioline
Summary: This is a collection of Bioline protocols for the SensiFAST™ Kits. Please refer to the appropriate protocol, depending on your application.
Proper citation: Bioline 2016. SensiFAST™ Protocols. protocols.io dx.doi.org/10.17504/protocols.io.gakbscw Copy
Authors: E. Dale Abel
Group: Diabetic Complications Consortium
Summary: Summary:This protocol describes the procedure used by the DiaComp for measuring left ventricular performance in isolated retrogradely perfused mouse hearts.Diabetic Complication:
Proper citation: E. Dale Abel 2019. Measurement of Left Ventricular Performance in Langendorff Perfused Mouse Hearts. protocols.io dx.doi.org/10.17504/protocols.io.7tfhnjn Copy
Authors: Shin Lin, Cole Trapnell, Jay Shendure
Summary: This document provides an overview of the protocols used by the Caltech-UW TMC to perform sci-RNA-seq2. This involves sequencing of the transcriptomes of dissociated single cells in a massively parallel manner.
Proper citation: Shin Lin, Cole Trapnell, Jay Shendure 2020. sci-RNA-seq2 Pipeline for the Human Biomolecular Atlas Program (HuBMAP). protocols.io dx.doi.org/10.17504/protocols.io.bqwamxae Copy
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