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Authors: Lora Starrs, Debra Brown, Sarah Popp, Charmaine Simeonovic
Summary: Paraffin sections (4 μm thickness) of formalin-fixed human pancreases were treated with heat/citrate buffer for antigen retrieval. Sdc-1core proteins were detected immunohistochemically using rat anti-mouse CD138 (Sdc-1) mAb (BD Biosciences), with horseradish peroxidase-conjugated rabbit anti-rat Ig (Dako). Background staining was checked using the corresponding isotype control Ig instead of the primary antibody. 3-amino-9-ethylcarbazole (AEC) was used as the chromogen. For morphometry, stained sections were imaged using a light microscope with attached camera (Olympus BX41). Image J software with color deconvolution plugin was used for the quantitative analysis of the % of islet area stained.

Proper citation: Lora Starrs, Debra Brown, Sarah Popp, Charmaine Simeonovic 2020. Immunohistochemical staining of Syndecan-1 (Sdc-1) core proteins in islet beta cells of formalin-fixed human pancreas. protocols.io https://dx.doi.org/10.17504/protocols.io.bmghk3t6 Copy   

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Authors: Don Cooper, Timothy Wandell, ben
Group: XPRIZE Rapid Covid Testing

Proper citation: Don Cooper, Timothy Wandell, ben 2020. NuFaction SARS-CoV2 Antigen Test Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bkv2kw8e Copy   

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Authors: Bulent Arman Aksoy, Pinar Aksoy, Elinor Gottschalk, Jeff Hammerbacher
Group: Hammer Lab
Summary: This protocol repurposes Promega's T Cell Activiation Bioassay workflow to be able to test relative mouse TCR reactivity against a cell line. This specific protocol uses MC38 as the target as it doesn't normally present SIINFEKL and have good H2Kb and H2Db expression levels. The reactivity will be in relative to the positive control (OT-I reactivity against SIINFEKL-pulsed cells) and the negative control (OT-I reactiviy against unpulsed cells).

Proper citation: Bulent Arman Aksoy, Pinar Aksoy, Elinor Gottschalk, Jeff Hammerbacher 2020. Measuring relative reactivity of mouse TCRs against a mouse cancer cell line. protocols.io https://dx.doi.org/10.17504/protocols.io.ba8gihtw Copy   

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Authors: Alyssa Alsante
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Van Etten, J. (n.d.). Formulation of Modified Bold's Basal Medium (MBBM). Retrieved from http://ncv.unl.edu/vanettenlab/

Proper citation: Alyssa Alsante 2016. MBBM Media. protocols.io https://dx.doi.org/10.17504/protocols.io.gdrbs56 Copy   

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Authors: Etienne Laliberté, Raymond Soffer
Group: Canadian Airborne Biodiversity Observatory
Summary: Here we describe the standardised protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure leaf spectral reflectance and transmittance, using the Spectra Vista Corporation (SVC) DC-R/T Integrating Sphere fitted to a portable full-range SVC HR-1024i field spectroradiometer. This standard version of our protocol describes the common case where an individual leaf is large enough to entirely cover the reflectance or transmission port of the integrating sphere. Briefly, six mature, healthy-looking and sunlit leaves from a canopy plant are selected for measurements of adaxial reflectance and transmittance. Reflectance measurements are referenced to a calibrated Spectralon® disk and corrected for stray light. Our leaf spectroscopy protocol builds from that of the Carnegie Airborne Observatory.

Proper citation: Etienne Laliberté, Raymond Soffer 2018. Measuring spectral reflectance and transmittance (350-2500 nm) of large leaves using the Spectra Vista Corporation (SVC) DC-R/T Integrating Sphere. protocols.io https://dx.doi.org/10.17504/protocols.io.p8pdrvn Copy   

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Authors: vggh
Summary: Validated with : rodent liver, kidney, eye, skeletal muscles, adrenal gland. 

Proper citation: vggh 2018. QIAGEN Allprep mini RNA extraction with QIAGEN RNase-Free DNase Set.. protocols.io https://dx.doi.org/10.17504/protocols.io.n62dhge Copy   

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Authors: Anibal Arce Medina
Group: Laboratorio de Tecnologias Libres

Proper citation: Anibal Arce Medina 2017. Step 2: Preparing amino acid, polyphosphates, and maltodextrin-based energy solutions for cell-free reactions. protocols.io https://dx.doi.org/10.17504/protocols.io.iefcbbn Copy   

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Authors: Zachariah Mclean, Sarah Appleby, Lisanne Fermin, Bjorn Oback
Summary: This protocol summarises zona-free sheep cloning by somatic cell transfer (SCT), adapted from a similar SCT method in cattle (https://doi.org/10.1089/153623003772032763). It describes the workflow that is required for reprogramming somatic cells into cloned blastocysts of high morphological quality, including two optional steps of embryo aggregation and vitrification. The resulting embryos can then be transferred into surrogate ewes for producing live cloned animals. Compared to conventional zona-intact procedures, this zona-free embryo reconstruction system increases throughput of cloned embryo production and ease of operation.

Proper citation: Zachariah Mclean, Sarah Appleby, Lisanne Fermin, Bjorn Oback 2020. Generating cloned sheep embryos by zona-free somatic cell transfer. protocols.io https://dx.doi.org/10.17504/protocols.io.begbjbsn Copy   

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Authors: Marie D., Le Gall F., Edern R., Gourvil P., Vaulot, D.
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G), Roscoff Culture Collection
Summary: This protocol can be used :to isolate novel cultures from natural samplesto isolate novel cultures from enriched samplesto purify existing cultures and remove contaminantsto obtain clonal cultures from a unialgal strainReference : Marie, D., Le Gall, F., Edern, R., Gourvil, P. & Vaulot, D. 2017. Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry. J. Phycol. in press.

Proper citation: Marie D., Le Gall F., Edern R., Gourvil P., Vaulot, D. 2018. Establishment of clonal algal cultures by flow cytometry sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.mn4c5gw Copy   

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Authors: Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire
Group: Human Cell Atlas Method Development Community, NCIHTAN, My protocols

Proper citation: Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire 2020. Copy of Cell preparation for scRNA-Seq from diluted bodily fluids. protocols.io https:// Copy   

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Authors: Contreras-Treviño HI, Reyna-Rosas E, Padilla-Noriega L.
Summary: The procedures to determine the half-lives of proteins by pulse-chase analysis and cycloheximide blocking are well established. In the case of oligomeric proteins, these methods do not differentiate whether the stabilities of the monomeric versus oligomeric forms of the protein differ. This method describes how to determine the half-lives of oligomeric proteins that are stable in the presence of low concentrations of the detergent SDS by combining cycloheximide blocking with non-dissociating Western blot (Dutta et al., 2011). The addition of cycloheximide inhibits protein synthesis and non-dissociating Western blot uses low concentrations of SDS to separate stable oligomers from monomers. The amount of the protein of interest detected in samples taken at different times after adding cycloheximide can be used to determine the half-life of the oligomeric protein.ReferencesByk, L., Iglesias, N., De Maio, F., Gebhard, L., Rossi, M. and Gamarnik, A. (2016). Dengue virus genome uncoating requires ubiquitination. mBio, 7(3), pp.e00804-16.Dutta, D., Chattopadhyay, S., Bagchi, P., Halder, U., Nandi, S., Mukherjee, A., Kobayashi, N., Taniguchi, K. and Chawla-Sarkar, M. (2011). Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3). Journal of Biological Chemistry, 286(22), pp. 20065-20077.Elroy-Stein, O., Moss, B., 2001. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system. In: Current Protocols in Molecular Biology, chapter 16, unit16 9.Ward, G.A., Stover, C.K., Moss, B., Fuerst, T.R., 1995. Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells. Proceding of the Nationall Academy of Sciences U S A, 92, pp. 6773-6777.

Proper citation: Contreras-Treviño HI, Reyna-Rosas E, Padilla-Noriega L. 2017. Determining half-lives of oligomeric proteins by cycloheximide blocking and non-dissociating Western blot. protocols.io https://dx.doi.org/10.17504/protocols.io.iiwccfe Copy   

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Authors: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: This real-time TaqMan-MGB RT-PCR protocol aimed to amplify measles virus (MeV) strains and not other viruses.Michael Lyon and Mitchell Finger designed the assay in 2009 using Primer Express software.The method was later published by Greg Smith in 2010 (see below).The assay targets the fusion (F) gene region and is designed as a qualitative test for investigating MeV infection of humans.This was a past assay that we no longer in use. For our favoured Measles virus TaqMan test, please refer to the MeV N TaqMan protocol.

Proper citation: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay 2018. Measles virus TaqMan RT-PCR (no longer in regular use; see Guidelines). protocols.io https://dx.doi.org/10.17504/protocols.io.rs6d6he Copy   

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Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: Published work using this protocol:- Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes.Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. PMN- 05 Culture of Human PMN - IL-8 production. protocols.io https://dx.doi.org/10.17504/protocols.io.bpx3mpqn Copy   

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Authors: François-Yves Bouget, Valérie Vergé and Jean-Claude Lozano
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes the preparation of cells and introduction of DNA into the cells by electroporation.  For selection of stable transformants or measure of transient gene expression see related protocols.

Proper citation: François-Yves Bouget, Valérie Vergé and Jean-Claude Lozano 2017. Transient transformation of Ostreococcus species (OTTH595, RCC809 and RCC802) and Bathycoccus. protocols.io https://dx.doi.org/10.17504/protocols.io.g86bzze Copy   

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Authors: don.cooper
Group: Mobile Assay, COVIDScanDx

Proper citation: don.cooper 2020. COVIDscanDX mScanner Protocol (Confidential). protocols.io https://dx.doi.org/10.17504/protocols.io.bkyckxsw Copy   

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Authors: Nicholas Leigh
Summary: This protocol is designed to obtain blastema cells (and wound epidermis if left attached) into a single cell suspension. It was specifically designed for single cell RNA sequencing, but can be used for other applications in which a single cell suspension is desired. The goal is to obtain a single cell suspension with near 100% viability in a rapid fashion. This protocol does not use centrifugation, but instead uses a gradient replacement strategy pioneered by Briggs et al, 2018 Science. This strategy washes away free-floating RNA from cells by using progressively denser solutions to "lift" off the solution in which the cells are currently emersed. This protocol could be can be modified to utilize centrifugation instead and likely should be if planning on anything other than single cell RNAseq on inDrops as a next step.

Proper citation: Nicholas Leigh 2018. Axolotl blastema dissociation into single cell suspension. protocols.io https://dx.doi.org/10.17504/protocols.io.qmmdu46 Copy   

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Authors: Aditya Mohan

Proper citation: Aditya Mohan 2019. CRISPR RNP Electroporation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8yfhxtn Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]

Proper citation: Angel Justiz-Vaillant 2020. Enzyme linked immunosorbent assay (ELISA) for determining the plasma or serum concentration of IL-33 in humans.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkexktfn Copy   

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Authors: Peng Wang, Hongjian Ni, Ruihe Wang

Proper citation: Peng Wang, Hongjian Ni, Ruihe Wang 2018. Fig 2. Forces of drill-string micro-element. protocols.io https://dx.doi.org/10.17504/protocols.io.ke2ctge Copy   

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Authors: Imen Lassadi
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Imen Lassadi 2017. Fluorescence activated cell sorting (FACS) of Perkinsus marinus transformants. protocols.io https://dx.doi.org/10.17504/protocols.io.hh2b38e Copy   

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