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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are either selected or depleted by incubating the 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. If these are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.Sample Preparation:Enzymatic digestion of mouse brain followed by myelin removal is recommended to achieve the highest purity and yield. There are several protocols published that can be applied. As a general guideline, Trypsin digestion followed by a 70/37/30% percoll gradient will increase final purity and yield.Scale up volumes if using 14 ml tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Selection Kits Protocol - 5. protocols.io dx.doi.org/10.17504/protocols.io.7z9hp96 Copy   

•   Source: Protocols.io

Authors: Liting Guo: Department of Endocrinology

Proper citation: Liting Guo: Department of Endocrinology 2017. Effects of Serum 25-Hydroxyvitamin D Level on Decreased Bone Mineral Density at Femoral Neck and Total Hip in Chinese Type 2 Diabetes. protocols.io dx.doi.org/10.17504/protocols.io.kjycupw Copy   

•   Source: Protocols.io

Authors: Kerry Wiles
Group: Laboratory of Systems Pharmacology, NCIHTAN
Summary: The biosafety guidelines in this protocol are based on the Center for Disease Control's Biosafety in Microbiology and Biomedical Laboratories (BMBL), which has served as the cornerstone of biosafety practice in the United States since its initial release in 1984. The information in this protocol was extracted from the BMBL and truncated to provide CHTN VUMC staff members with the knowledge necessary to complete their training requirements. The protocol is used as both a reference and training SOP for the tissue procurement and the bodily fluids/blood processing protocols.

Proper citation: Kerry Wiles 2021. Tissue Procurement: Biosafety Guidelines. protocols.io dx.doi.org/10.17504/protocols.io.6y6hfze Copy   

•   Source: Protocols.io

Authors: Alan Cone
Group: Ju Lab

Proper citation: Alan Cone 2016. Destain Buffer. protocols.io dx.doi.org/10.17504/protocols.io.fw9bph6 Copy   

•   Source: Protocols.io

Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. Th9, Th2, Th17, mast cells, NKT cells, and regulatory T cells produce interleukin-9. It enhances T-cell survival, mast cell activation and synergy with erythropoietin.[1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-9 in human serum or plasma.. protocols.io dx.doi.org/10.17504/protocols.io.bkevkte6 Copy   

•   Source: Protocols.io

Authors: Tue Sparholt Jørgensen

Proper citation: Tue Sparholt Jørgensen 2021. Sanger sequencing of a part of the SARS-CoV-2 spike protein. protocols.io dx.doi.org/10.17504/protocols.io.bsbdnai6 Copy   

•   Source: Protocols.io

Authors: Promega, Trevor Wagner
Group: Promega
Summary: GoTaq® qPCR Master Mix(a,b) is a reagent system for quantitative PCR (qPCR). The system contains a new fluorescent DNA-binding dye that often exhibits greater fluorescence enhancement upon binding to double-stranded DNA (dsDNA) than SYBR. Green I.GoTaq® qPCR Master Mix is provided as a simple-to-use, stabilized 2X formulation that includes all components for qPCR except sample DNA, primers and water. This formulation, which includes a proprietary dsDNA-binding dye, a low level of carboxy-X-rhodamine (CXR) reference dye (identical to ROX™ dye), GoTaq. Hot Start Polymerase, MgCl2, dNTPs and a proprietary reaction buffer, produces optimal results in qPCR experiments. A separate tube of CXR Reference Dye is included for use with instruments that require a higher level of reference dye than that in the GoTaq® qPCR Master Mix.Advantages of the GoTaq® qPCR Master MixDye: The proprietary dye provides brighter dsDNA-dependent fluorescence than SYBR. Green I, with less PCR inhibition than SYBR® Green. The dye enables efficient amplification, resulting in earlier quantification cycle (Cq) values and an expanded linear range using the same filters and settings as SYBR® Green I. The CXR reference dye can be detected using the same filters and settings as those used for ROX™ dye.Quantification cycle is formerly known as cycle threshold (Ct).Polymerase/Buffer Formulation: GoTaq® Hot Start Polymerase contains full-length Taq DNA polymerase bound to a proprietary antibody that prevents polymerase activity at room temperature. Thermal activation is achieved by incubating the assembled reaction at 95°C for 2 minutes. The proprietary polymerase/buffer formulation accommodates extended cycle numbers (45–50 cycles) and is compatible with thermal cycling programs that require extended activation (95°C for 10 minutes).Performance: You can expect reliable performance with minimal lot-to-lot variation: efficient, sensitive and linear qPCR amplification over a wide dynamic range.GoTaq® qPCR Master Mix ProtocolIf you are currently performing dye-based qPCR, the GoTaq® qPCR Master Mix can simply be substituted for your current master mix. For consistency within an experimental set, prepare a sufficient volume of reaction mix without template DNA for the DNA standard reactions and experimental sample reactions. The protocol for a 50μl reaction is outlined below. Component volumes may be scaled as appropriate. This protocol assumes that 20% of the reaction volume is DNA template (e.g., 10μl of DNA template added to 40μl of reaction mix). If the volume of DNA template is more or less than 10μl, adjust the volume of Nuclease-Free Water accordingly so that the final reaction volume is 50μl.

Proper citation: Promega, Trevor Wagner 2018. How to Setup and Perform a qPCR Experiment.. protocols.io dx.doi.org/10.17504/protocols.io.k5jcy4n Copy   

•   Source: Protocols.io

Authors: Caroline Grabbe, Ingrid Dacklin, Behzad Khoshnood

Proper citation: Caroline Grabbe, Ingrid Dacklin, Behzad Khoshnood 2017. Batch absorption of Urm1-Flag conjugates from Drosophila tissues for mass spectrometry analysis. protocols.io dx.doi.org/10.17504/protocols.io.jizckf6 Copy   

•   Source: Protocols.io

Authors: Laura Poza-Viejo, Ivan del Olmo, Pedro Crevillén
Summary: Chromatin Immunoprecipitation (ChIP) is a crucial technique to study chromatin regulation, epigenetic phenomena and transcription factor DNA-binding in vivo. This technique is widely used in model plant systems like Arabidopsis but reliability in other plant systems is challenging. We adapted our well-established Arabidopsis ChIP protocol to be used with leaves and inflorescences of Brassica crops species (Brassica rapa and Brassica napus). This protocol was originally based on Gendrel et al. Nature Methods volume 2, pages 213–218 (2005), but has several modifications inlcluding chromatin sonication using a BioRuptor (Diagenode), immunoprecipitation using magnetic Dynabeads (Thermofisher) and DNA elution recovery performed using Chelex 100 (Bio Rad). The protocol is quick and very reproducible and has been validated against a number of histone modifications and protein tag antibodies. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Laura Poza-Viejo, Ivan del Olmo, Pedro Crevillén 2019. Plant Chromatin Immunoprecipitation. protocols.io dx.doi.org/10.17504/protocols.io.zmff43n Copy   

•   Source: Protocols.io

Authors: David O'Connor, Natan Vega Potler, Meagan Kovacs, Ting Xu, Lei Ai, John Pellman, Tamara Vanderwal, Lucas Parra, Samantha Cohen, Satrajit Ghosh, Jasmine Escalera, Natalie Grant-Villegas, Yael Osman, Anastasia Bui, R Cameron Craddock, Michael P Milham
Group: GigaScience Press
Summary: This protocol describes MRI, voice recoding, and some basic physiological measurements and questionnaires for Sessions 2-7 and 9-14 of the following work:David O'Connor, et. al. (2017) The Healthy Brain Network Serial Scanning Initiative. GigaScience...

Proper citation: David O'Connor, Natan Vega Potler, Meagan Kovacs, Ting Xu, Lei Ai, John Pellman, Tamara Vanderwal, Lucas Parra, Samantha Cohen, Satrajit Ghosh, Jasmine Escalera, Natalie Grant-Villegas, Yael Osman, Anastasia Bui, R Cameron Craddock, Michael P Milham 2017. The Healthy Brain Network Serial Scanning Initiative, Sessions 2-7 and 9-14. protocols.io dx.doi.org/10.17504/protocols.io.gxubxnw Copy   

•   Source: Protocols.io

Authors: Magdalena Julkowska
Group: Salt Lab KAUST
Summary: Protocol adapted from Shin et al., 2005 and Orman-Ligeza et al. 2016 (citing Shin)

Proper citation: Magdalena Julkowska 2020. ROS staining. protocols.io dx.doi.org/10.17504/protocols.io.peidjce Copy   

•   Source: Protocols.io

Authors: Gwendolyn Gallagher
Group: Coleman Lab
Summary: Purpose: Preparation of culture samples for mass spectrometry-based proteomicsPrinciple: Utilizing a membrane-enrichment method of lysing cells and preparing peptides has yielded higher representation of membrane proteins in our mass spectrometry-based proteomic results.Traditional methods do not adequately extract or digest hydrophobic, transmembrane proteins. Particularly, we can now see full expression patterns of proteorhodopsin, something we could not detect using traditional mass spec proteomics prep.This protocol builds on the work of Molloy (2008) Methods Mol Biol (doi:10.1007/978-1-60327-064-9_30), Erde et al. (2014) J. Proteome Res. (doi:10.1021/pr4010019), and Waldbauer, et al. (2017) Anal. Chem. (doi: 10.1021/acs.analchem.7b02752).Pure culture samples were spun down and flash frozen for proteomics.A carbonate extraction protocol was used for membrane enrichment before eFASP.The membrane fraction was enzymatically digested with both chymotrypsin and trypsin and the cytosolic fraction was digested with just trypsin.These samples were then ready to be processed further by in vitroisotopic peptide labeling (diDO-IPTL).

Proper citation: Gwendolyn Gallagher 2020. A membrane-enriched preparation of culture samples for mass spectrometry-based proteomics. protocols.io dx.doi.org/10.17504/protocols.io.bdapi2dn Copy   

•   Source: Protocols.io

Authors: Hansheng Zhao

Proper citation: Hansheng Zhao 2018. Genome-wide identification of genes involved in the lignin biosynthetic pathway. protocols.io dx.doi.org/10.17504/protocols.io.phjdj4n Copy   

•   Source: Protocols.io

Authors: Lu Wang, Brittany Sprecher, Huan Zhang, and Senjie Lin
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Lu Wang, Brittany Sprecher, Huan Zhang, and Senjie Lin 2019. Co-Incubation protocol for transforming heterotrophic dinoflagellates (e.g. Oxyrrhis marina). protocols.io dx.doi.org/10.17504/protocols.io.7pphmmn Copy   

•   Source: Protocols.io

Authors: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie
Group: Coronavirus Method Development Community, XPRIZE Rapid Covid Testing
Summary: SalivaDirect is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets.Version 2 includes: Optimized thermocycler conditionsLocally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclersUse of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates.Version 3 has been updated to remove steps for sample self-collection.Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx.

Proper citation: Chantal Vogels, Doug E. Brackney, Chaney Kalinich, Isabel Ott, Nathan Grubaugh, Anne Wyllie 2020. SalivaDirect: RNA extraction-free SARS-CoV-2 diagnostics. protocols.io dx.doi.org/10.17504/protocols.io.bjswknfe Copy   

•   Source: Protocols.io

Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: List of published works using this protocol:- Kustrimovic N., Comi C., Magistrelli L., Rasini E., Legnaro M., Bombelli R., Aleksic I., Blandini F., Minafra B., Riboldazzi G., Struchio A., Mauri M., Bono G., Marino F., Cosentino M. Parkinson’s disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients (2018). Journal of neuroinflammation, 15(1), 205. https://doi.org/10.1186/s12974-018-1248-8- Kustrimovic, N., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Comi, C., Mauri, M., Minafra, B., Riboldazzi, G., Sanchez-Guajardo, V., Marino, F., & Cosentino, M. (2016). Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson's Disease. Scientific reports, 6, 33738. https://doi.org/10.1038/srep33738- Cosentino M., Ferrari M., Kustrimovic N., Rasini E., Marino F. (2015). Influence of dopamine receptor gene polymorphisms on circulating T lymphocytes: A pilot study in healthy subjects. Human immunology, 76, 10, 747-752. https://doi.org/10.1016/j.humimm.2015.09.032

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PBMC- 02 - CD4+ T cell Isolation from PBMC with “Dynabeads CD4 Positive Isolation Kit”. protocols.io dx.doi.org/10.17504/protocols.io.bpxqmpmw Copy   

•   Source: Protocols.io

Authors: Yaowu Yuan
Group: Mimulus
Summary: This collection consists of three protocols: 1. Agro Transformation a) Using Electropotator b) Using Freeze-Thaw Method2. Agro Preparation 3. Plant infiltration

Proper citation: Yaowu Yuan 2019. Mimulus in planta Transformation. protocols.io dx.doi.org/10.17504/protocols.io.3tkgnkw Copy   

•   Source: Protocols.io

Authors: RACHELE CESARONI, Rachele Cesaroni
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: RACHELE CESARONI, Rachele Cesaroni 2019. Euplotes crassus transfection using Lipofectamine 2000 as vehicle (provisional). protocols.io dx.doi.org/10.17504/protocols.io.2aigace Copy   

•   Source: Protocols.io

Authors: Brenton Paolella
Summary: This protocol is for performing Cell Hashing only. Sample multiplexing and super-loading on single cell RNA-sequencing platforms.Cell Hashing uses a series of oligo-tagged antibodies against ubiquitously expressed surface proteins with different barcodes to uniquely label cells from distinct samples, which can be subsequently pooled in one scRNA-seq run. By sequencing these tags alongside the cellular transcriptome, we can assign each cell to its sample of origin, and robustly identify doublets originating from multiple samples.

Proper citation: Brenton Paolella 2019. Cell Hashing. protocols.io dx.doi.org/10.17504/protocols.io.yp6fvre Copy   

•   Source: Protocols.io

Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using T4 DNA Polymerase.

Proper citation: New England Biolabs 2015. Blunting Protocol (M0203). protocols.io dx.doi.org/10.17504/protocols.io.cgwtxd Copy   

•   Source: Protocols.io