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Authors: Lance Vowell
Group: Workshop: FSCI Research Reproducibility in Theory and Practice
Summary: FSCI Reproducibilty workshop
Proper citation: Lance Vowell 2018. Lance's Protocol. protocols.io dx.doi.org/10.17504/protocols.io.r7yd9pw Copy
Authors: Tatsuya Suwabe, Francisco J. Barrera-Flores, Rene Rodriguez-Gutierrez, Yoshifumi Ubara, Kenmei Takaichi
Summary: End-stage renal disease (ESRD) is related to high morbidity, mortality, and impaired health-related quality of life. While hemodialysis (HD) is the current life-saving standard of treatment for patients with ESRD, their quality of life (QoL) remains far from desirable. Online HDF (OL-HDF), due to its convenience, could improve the QoL of patients with ESRD, however, this remains uncertain.We will aim to assess the body of evidence of OL-HDF compared to HD regarding QoL in patients with ESRD.We will comprehensively search in MEDLINE, EMBASE, The Cochrane Database of Systematic Reviews, and The Cochrane Central Register of Controlled Trials databases from their inception to February 2018. Reviewers working independently and in duplicate appraised the quality and included randomized controlled trials (RCTs) that evaluated, in patients with ESRD and HD or OL-HDF, QoL (Short Form Health Survey with 36 questions (SF-36) with physical component score (PCS) and mental component score (MCS) as well as scores about social activity, fatigue, and emotion). A meta-analysis of each outcome of interest will be performed using a random-effects model.
Proper citation: Tatsuya Suwabe, Francisco J. Barrera-Flores, Rene Rodriguez-Gutierrez, Yoshifumi Ubara, Kenmei Takaichi 2018. Effect of Online Hemodiafiltration compared with Hemodialysis on Quality of Life in patients with ESRD: A Systematic Review and Meta-analysis of Randomized Trials. protocols.io dx.doi.org/10.17504/protocols.io.rhnd35e Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.
Proper citation: Sam Li 2019. MojoSort™ Mouse Neutrophil Isolation Kit Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7yuhpww Copy
Authors: Maria Rubio-Brotons
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab
Proper citation: Maria Rubio-Brotons 2016. Super simple In vivo Hoechst staining of unicellular protists. protocols.io dx.doi.org/10.17504/protocols.io.gg6btze Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Human iPSCs are an ideal system for studying human biology due to their rapid proliferation, genomic stability, and ability to differentiate into many somatic cell types. Historically, specialized culture practices and costly reagents have hindered widespread adoption of iPSCs by the cell biology community. In recent years, however, development of new culture techniques and improved media formulations have dramatically simplified iPSC culture and reduced costs.The protocols described in this unit are adapted from a collection of publications that establish optimal practices for the maintenance of human iPSC cultures (Beers et al., 2012; Ludwig et al., 2006; Chen et al., 2011). While these publications provide useful guidelines for the stem cell novice, here we distill the fundamental procedures necessary for maintaining iPSCs in a pluripotent state and highlight critical steps that may need to be optimized for individual applications. In practice, iPSC lines of interest are usually maintained in an undifferentiated state in small cultures (1 to 3 wells of a 6-well plate) to reduce reagent use before being expanded as needed for experimentation.Essential 8 (E8) is the simplest defined medium for hiPSC culture. E8 may be prepared from its components by the consumer (Table 1; Chen et al., 2011) or purchased as a preformulated kit. Other commercially available media may be substituted, such as mTeSR1 or StemFlex. A volume of 12 ml of medium should be added to each 10-cm tissue culture dish or distributed evenly across each standard multiwell plate (i.e., 2 ml/well for a 6-well plate). E8 should be aspirated and replaced with fresh medium daily, although a double volume may be added at low confluency to permit an extra day of culture without medium changes. StemFlex and E8 Flex contain components that stabilize the recombinant growth factors present in the medium, permitting medium exchange every other day as a general practice. Some iPSC lines (e.g., WTC11) tolerate every-other-day medium changes of standard E8 medium without loss of pluripotency or cell death, further reducing costs of medium and consumables. Finally, mTeSR1 may promote cell survival in stressful conditions better than E8, especially for finicky iPSC lines, although supplementation with a ROCK inhibitor (RI) is also recommended in such scenarios. Use of standard E8 will be assumed throughout this basic protocol.Additionally, this protocol uses Matrigel-coated tissue culture plates. Matrigel works well for iPSC culture and has been widely adopted for research applications. However, since Matrigel is derived from murine sarcoma cells, it is not chemically defined and exhibits batch-to-batch variability. Alternative defined coatings include recombinant laminin or vitronectin, although these substrates are typically more costly. Notably, downstream neural differentiation described in these protocols occurs in fully defined conditions (see Basic Protocols 5 to 8), so the choice of iPSC substrate is of minimal scientific importance to all but clinical-grade applications.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. MAINTENANCE CULTURE OF iPSCs (Basic Protocol 1). protocols.io dx.doi.org/10.17504/protocols.io.48vgzw6 Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.
Proper citation: Sam Li 2019. MojoSort™ Human NK Cell Isolation Kit Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7ythpwn Copy
Authors: Jennifer Brum
Group: VERVE Net, Sullivan Lab
Summary: Purpose: This protocol describes how to adsorb viruses onto TEM (transmission electron microscopy) grids. The sample is allowed to sit on a hydrophilic grid and viruses adsorb onto the surface of the grid. This technique is generally used for viral lysates with high concentrations of viruses. For natural samples, use the protocol “Quantitatively Depositing Viruses onto TEM Grids using an Airfuge”.Note: If purification of the viruses is necessary, refer to Ackermann and Heldal (2010) for various options.
Proper citation: Jennifer Brum 2016. Adsorbing Viruses on TEM Grids. protocols.io dx.doi.org/10.17504/protocols.io.dar2d5 Copy
Authors: Michiyo Tsuru
Group: Clinical Proteomics and Gene Therapy Laboratory
Summary: Proteins from the spinal ligament cells of wild-type and null mice were separated using two-dimensional gel electrophoresis and stained with Pro-Q Diamond phosphoprotein gel stain (Thermo Fisher Scientific, Waltham, MA, USA) followed by SYPRO Ruby protein gel stain (Thermo Fisher Scientific). The gel was visualized by using a Molecular Imager FX Pro Plus multi imager system (Bio-Rad, Hercules, CA, USA), and the images were acquired using PDQuest software, version 8.0 (Bio-Rad). The composite images were digitally pseudo-colored and overlaid. The gels were treated with a phosphorylation reagent, and the phosphorylation spots were verified using LC–MS/MS after in-gel digestion using trypsin and peptide extraction, which were performed according to a previously published protocol. Purified peptides (20–30 pmol) were analyzed by using the UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to an Orbitrap Elite linear ion trap mass spectrometer (Thermo Fisher Scientific) with an in-house manufactured nano-electrospray ionization interface. For micro reversed-phase LC-MS/MS analysis, the samples were injected into a trap column (nano; 75 × 280 μm inner × outer diameter; packed with 15 cm Acclaim Pep Map C18). Buffer A (0.1 % formamide) and Buffer B (80 % acetonitrile and 0.0 8% formic acid) were used to elute the bound peptides with a split flow system (flow rate: 300 nL/min) for 60 min on a linear gradient. In positive ion mode, spectra were acquired with cycles of one full MS scan in the linear trap quadrupole (m/z 350–2000) followed by 20 data-dependent MS/MS scans with a normalized collision energy of 35 %.
Proper citation: Michiyo Tsuru 2018. Phosphoproteomics. protocols.io dx.doi.org/10.17504/protocols.io.j9jcr4n Copy
Authors: Alba Balletbó
Group: iGEM Wageningen 2019
Summary: Protocol adapted from the NanoDrop 1000 Spectrophotometer V3.8 User’s Manual.
Proper citation: Alba Balletbó 2019. DNA Concentration Measurement (Protocol for Thermo Scientific NanoDrop™ 1000 Spectrophotometer). protocols.io dx.doi.org/10.17504/protocols.io.79khr4w Copy
Authors: Nancy Smith, Mourad Ferdaoussi, Haopeng Lin, Patrick Macdonald
Group: CIRTNR2FIC
Summary: This protocol details the insulin tolerance test in mice. This is performed on C57Bl6, transgenic SENP1 KO and ZMIZ1 KO mouse models. Typically glucose tolerance tests (GTTs) are performed at 12 weeks of age, followed by an ITT 2 weeks later. Some mice are put on High Fat Diet and an ITT repeated following 10 weeks on High Fat Diet.Once the in vivo studies are complete, the pancreas or islets are isolated for further ex vivo experiments.
Proper citation: Nancy Smith, Mourad Ferdaoussi, Haopeng Lin, Patrick Macdonald 2020. Insulin Tolerance Test in Mouse. protocols.io dx.doi.org/10.17504/protocols.io.wxjffkn Copy
Authors: Carlotta Catozzi, Anna Cusco
Summary: This workflow was performed using QIIME2 for a longitudinal study. The aim was to investigate the changes in mcirobiota after antibiotic and Lactobacillus rhamnosus treatment in water buffaloes affected by subclinical mastitis.
Proper citation: Carlotta Catozzi, Anna Cusco 2019. Qiime 2 Workflow - Longitudinal study. protocols.io dx.doi.org/10.17504/protocols.io.ucpesvn Copy
Authors: Samuel Nahashon
Summary: Probiotics are live microbial feed supplements that promote growth and health to the host by minimizing non-essential and pathogenic microorganisms in the host’s gastrointestinal tract (GIT). The campaign to minimize excessive use of antibiotics in poultry production has necessitated development of probiotics with broad application in multiple poultry species. Design of such probiotics requires understanding of the diversity or similarity in microbial profiles among avian species of economic importance. Therefore, the objective of this research was to establish and compare the microbial profiles of the GIT of Guinea fowl and chicken and to establish the microbial diversity or similarity between the two avian species. A metagenomic approach consisting of the amplification and sequence analysis of the hypervariable regions V1-V9 of the 16S rRNA gene was used to identify the GIT microbes. Collectively, we detected more than 150 microbial families. The total number of microbial species detected in the chicken GIT was higher than that found in the Guinea Fowl GIT. Our studies also revealed phylogenetic diversity among the microbial species found in chicken and guinea fowl. The phylum Firmicutes was most abundant in both avian species whereas Phylum Actinobacteria was most abundant in chickens than Guinea fowls. The diversity of the microbial profiles found in broiler chickens and Guinea fowls suggest that the design of effective avian probiotics would require species specificity.
Proper citation: Samuel Nahashon 2017. Metagenomic Analysis of Microbial profile of Guinea Fowls and Chickens. protocols.io dx.doi.org/10.17504/protocols.io.mdgc23w Copy
Authors: Bonnie Poulos
Group: VERVE Net, Sullivan Lab
Proper citation: Bonnie Poulos 2016. Transcriptomics During One-Step Growth Curves for Cellulophaga Phages. protocols.io dx.doi.org/10.17504/protocols.io.ek4bcyw Copy
Authors: N.J. Hillson
Proper citation: N.J. Hillson 2019. Gibson Protocol. protocols.io dx.doi.org/10.17504/protocols.io.6t5heq6 Copy
Authors: Nawel Zaibi; Pengyun Li; Shang-Zhong Xu
Group: Hull York Medical School (Dr Xu Lab), Hull York Medical School
Summary: This protocol is for the detection of cytosolic and mitochondrial ROS using FlexStation 3
Proper citation: Nawel Zaibi; Pengyun Li; Shang-Zhong Xu 2021. Detection of cytosolic and mitochondrial ROS production using FlexStation 3. protocols.io dx.doi.org/10.17504/protocols.io.bsb2naqe Copy
Authors: Anna Behle
Group: Axmann Lab, CyanoWorld, iGEM Duesseldorf 2018
Summary: This protocol summarizes the process of primer design for assembly-based cloning methods, such as Gibson or AQUA-cloning. Well-designed primers are necessary for efficient cloning of any kind. This protocol can also be used to design primers for overlap extension PCR of linear fragments. Alternatively, overhangs can also include restriction sites for restriction/ligation based cloning.
Proper citation: Anna Behle 2016. Primer design for assembly-based cloning. protocols.io dx.doi.org/10.17504/protocols.io.gs4bwgw Copy
Authors: Alba Balletbó
Group: iGEM Wageningen 2019
Summary: The final step in the construction of a recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly digested vector backbone. This is accomplished by covalently connecting the sugar backbone of the two DNA fragments. This reaction, called ligation, is performed by the T4 DNA ligase enzyme. The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells for propagation.The majority of ligation reactions involve DNA fragments that have been generated by restriction enzyme ligation (See Protocols). Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results in a single stranded overhang on the digested end of the DNA fragment. The overhangs, called "sticky ends", are what allow the vector and insert to bind to each other. When the sticky ends are compatible, meaning that the overhanging base pairs on the vector and insert are complementary, the two pieces of DNA connect and ultimately are fused by the ligation reaction.
Proper citation: Alba Balletbó 2019. Ligation Protocol with T4 DNA Ligase. protocols.io dx.doi.org/10.17504/protocols.io.7khhkt6 Copy
Authors: Henri Brunengraber
Group: Mouse Metabolic Phenotyping Centers, Metabolomics Protocols & Workflows
Summary: Total glycerol is determined by first hydrolyzing glycerides (in plasma or tissues) in 1 N KOHethanol + heat and then the hydrolysate extracted following acidification ~pH 1.0. A two phase extract is created using chloroform. The aqueous phase contains the total glycerol and the organic phase contains total fatty acids. An aliquot of aqueous phase in dried down by nitrogen gas and the concentration and/or 2H-labelled glycerol is then determined by GC-MS following conversion of glycerol to its triacetate derivative. Glucose assay involves extraction of glucose from blood, plasma or tissues and the concentration and/or 2H/13C-labelled glucose is also determined by GC-MS following conversion to its pentaacetate derivative. Since the derivatizing methods are the same for glucose as glycerol, the can be assayed from a single sample preparation under the same GC-MS conditions and single run-time.References: 1. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem.;284(10):6101-8 (2009)
Proper citation: Henri Brunengraber 2019. Case - Glycerol and Glucose Assays by GC-mass spectrometry. protocols.io dx.doi.org/10.17504/protocols.io.ydkfs4w Copy
Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:The use of total radical-trapping antioxidant parameter (TRAP) has recently been proposed to explore the antioxidant property of a plasma sample. This assay is a measure of oxidative stress in the animals. This protocol describes the procedure used by the DiaComp to measure TRAP.Diabetic Complications:
Proper citation: Eva Feldman 2019. TRAP Assay. protocols.io dx.doi.org/10.17504/protocols.io.3sqgndw Copy
Authors: Ezequiel G. Mogro, Nicolas Ambrosis, Mauricio Lozano
Summary: Insertion sequences (ISs) are small transposable elements composed only by a transposase and imperfect terminal inverted repeats, which have an important role in genome evolution and contribute to bacterial genome plasticity and adaptability. Bacterial strains from a same species usually present genome rearrangements and variation in the location of insertion sequences and other transposable elements, which might produce phenotypic variations, including antibiotic resistance and adaptation to vaccination strategies. We developed ISCompare to profile IS mobilization events in related bacterial strains. Here we present a comprehensive description on how to use ISCompare, and interpret the obtained results.Basic protocol: Automatic search of Differentially located Insertion Sequences (DLIS)Support protocol 1: Search for differentially located ISs using local filesSupport protocol 2: Using the shift mode to identify differentially located Group II introns
Proper citation: Ezequiel G. Mogro, Nicolas Ambrosis, Mauricio Lozano 2021. Finding insertion sequence mobilization events with ISCompare.. protocols.io dx.doi.org/10.17504/protocols.io.bst6nere Copy
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