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Authors: Joshua Welsh, Jennifer Jones
Group: Translational Nanobiology Section
Summary: This protocol outlines the steps required to acquire fluorescence reference material data for use with the FCMPASS software. This is one of a number of protocols in the pipeline for performing small particle calibration using the fcmpass software package.

Proper citation: Joshua Welsh, Jennifer Jones 2020. FCMPASS - Acquisition and gating of fluorescence reference materials. protocols.io https://dx.doi.org/10.17504/protocols.io.bjcpkivn Copy   

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Authors: Rengin Yamur Akbiyik

Proper citation: Rengin Yamur Akbiyik 2018. Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.sjwecpe Copy   

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Authors: Tobias Weise, Bettina Boettcher, Slavena Vylkova
Summary: Biofilm formation under shear flow conditions was monitored using the Bioflux1000 device (Fluxion Biosciences, Inc.). In short, Candida albicans overnight cultures were washed in pre-warmed RPMI medium. Cells were seeded for 2-5 sec from the outlet well into the channels of Bioflux1000 flow chambers, which were primed before with warm medium. The cells were allowed to adhere to the channels for 90 min without any flow, followed by removal of non-adherent cells by flowing fresh, pre-warmed RPMI medium for 5 sec. Shear flow was set for time series experiments over 24 h biofilm formation and images were captured every 20 min. Two channels were investigated in parallel having a 10 × magnification to allow a direct comparison between a mutant and a reference (wild type) strain. Image capturing and stacks to movies was performed using the MetaMorph® Software (Molecular Devices).Source material provided as AVI files was converted into single TIFF images as well as data frames containing meta data annotations. The individual image contains two growth chambers (wild type and mutant) separated by four edge lines. Images were rotated automatically to vertical alignment in order to carry out an automated chamber detection and analysis. The mean pixel intensity (i. e. grey scale value; reflecting cell density) of the individual chamber was calculated and added into the respective data frame. An ODE model reflecting the logistic growth as well as the lag phase was fitted to the individual experiments. Fitting was carried out by minimising a cost function (unweighted least-squares-based) using the Nelder-Mead algorithm. Growth rate time series generated from the fitted model were used to compare wild type and mutant regarding the maximum observed growth rates at their respective time points.All computations were performed using the programming language python (version 3.6.9) and the additional packages numpy (version 1.16.2), opencv-python (version 4.1.1.26), pandas (version 0.25.0), scipy (version 1.3.1) and scikit-image (version 0.15.0).

Proper citation: Tobias Weise, Bettina Boettcher, Slavena Vylkova 2020. Bioflux Analyses. protocols.io https://dx.doi.org/10.17504/protocols.io.bb7qirmw Copy   

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Authors: Bianca Hemmeryckx, Dries Bauters, H. Roger Lijnen
Summary: Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (UCP-1) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration. Also in WAT adaptive UCP-1 positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT. This browning of WAT is enhanced by exposure to cold temperatures.

Proper citation: Bianca Hemmeryckx, Dries Bauters, H. Roger Lijnen 2018. UCP-1 (Abcam ab10983) immunohistochemical protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j9scr6e Copy   

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Authors: Gloria Pryhuber
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: Purpose and Scope of the ProcedureStandardize process for receiving lung donations Scope: Coordination of screening, acceptance and receipt of tissue for research programsPrinciples Lung donations rejected for transplantation can be used to understand human lung structure and development, ultimately to improve care of patients with lung diseaseRapid, standardized and safe processing of human tissue for the HTC requires coordination of a team of staff, materials and attention to protocolsRapid processing is required to maintain the tissues in a state as close to normal as possibleUltra-high resolution CT Scan in an inflated state will provide a high-level comparative assessment of human lung structure across developmental ages (See Protocol 603)Principles Lung donations rejected for transplantation can be used to understand human lung structure and development, ultimately to improve care of patients with lung diseaseRapid, standardized and safe processing of human tissue for the HTC requires coordination of a team of staff, materials and attention to protocolsRapid processing is required to maintain the tissues in a state as close to normal as possibleUltra-high resolution CT Scan in an inflated state will provide a high-level comparative assessment of human lung structure across developmental ages (See Protocol 603)

Proper citation: Gloria Pryhuber 2020. 602.2 Donor Acceptance Criteria for URMC HTC HuBMAP and LungMAP Inclusion. protocols.io https://dx.doi.org/10.17504/protocols.io.bjuxknxn Copy   

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Authors:
Group: UCSC BME 22L

Proper citation: 2020. Protocols for PCR. protocols.io https:// Copy   

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Authors: Sierra Simpson, Olivier George
Group: George Lab

Proper citation: Sierra Simpson, Olivier George 2020. AAV production for Serotypes with Heparin Binding Capabilities. protocols.io https:// Copy   

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Authors: Sarah Popp, Sarita Dhounchak, Charmaine Simeonovic
Summary: Isolated mouse islets were dispersed into single cells using Accutase (Millipore; 250 µl/500 islets). 4-8 x 104 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. Isolated mouse islet cells were cultured in the presence or absence of the HS mimetics heparin (a highly sulfated HS analogue from porcine intestinal mucosa) or PI-88 (Progen Pharmaceuticals Limited,) at 50 mg/ml for 2 days in 5% CO2, 95% air at 37ºC. In some studies islet cells were acutely treated with 30% H2O2 (Chem-Supply) as a source of reactive oxygen species (ROS) for 5 min on day 0 or after culture for 2 days with/without HS mimetics. Damaged and dying islet cells were assessed using Calcein-AM (Calcein; 0.04 µM; Invitrogen)/Propidium iodide (PI; 2.5 µg/ml; BD Biosciences) or by Sytox green (31.25 nmol/L; Invitrogen, Molecular Probes) uptake. BD LSR Fortessa flow cytometer BD FACS DIVA software (version 8) were used to collect events and Flow Jo software (version 10.0.7, TreeStar Inc.) was used to analyse the intensity of fluorescence staining.

Proper citation: Sarah Popp, Sarita Dhounchak, Charmaine Simeonovic 2020. Isolation of mouse islet cells, culture with heparan sulfate mimetics and flow cytometry analysis of beta cell viability. protocols.io https://dx.doi.org/10.17504/protocols.io.bmgjk3un Copy   

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Authors: 张 雪
Group: Zhang Xue

Proper citation: 张 雪 2020. lysis buffer裂解基因组. protocols.io https:// Copy   

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Authors: Mark Dewitt & Julia Wong
Group: Innovative Genomics Institute, CornLab
Summary: In vitro T7 template synthesis and transcriptionsgRNA protocol with SPRI beads

Proper citation: Mark Dewitt & Julia Wong 2015. In vitro transcription of guide RNAs. protocols.io https://dx.doi.org/10.17504/protocols.io.dm749m Copy   

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Authors: Irfan Uddin, Taeeb Ahmad, Furqan Aziz
Summary: This algorithm implements Reversible Data Hiding (RDH) technique by rearranging the columns (or rows) of the image in a way that enhances the smooth regions of an image. Any difference based technique to embed data can then be used in the transformed image.

Proper citation: Irfan Uddin, Taeeb Ahmad, Furqan Aziz 2020. RDH. protocols.io https://dx.doi.org/10.17504/protocols.io.bdyzi7x6 Copy   

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Authors: Angel Justiz-Vaillant
Summary: Protein LAG (SpLAG) is an immunoglobulin-binding protein that interacts with the Fc and Fab regions of many mammalian immunoglobulins. It is produced by a chemical coupling of individual proteins and them mixing it up to the appropriate protein ratio. SpLAG binds well to some avian immunoglobulin [1].References1.Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175

Proper citation: Angel Justiz-Vaillant 2020. Copy of Direct ELISA for investigating the binding of chemically-made Protein-LAG  to immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjxxkppn Copy   

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Authors: Dennis Dienst
Group: CyanoWorld

Proper citation: Dennis Dienst 2017. TSS Transformation (Chung et al., 1989, PNAS 86:2172-2175). protocols.io https://dx.doi.org/10.17504/protocols.io.jn5cmg6 Copy   

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Authors: Zbigniew Mikulski
Group: La Jolla Institute Microscopy Core
Summary: There is a wealth of microscopy-related information online. This document lists the ones we know and like. Click on the document tab to see the list!  

Proper citation: Zbigniew Mikulski 2018. Online microscopy and histology resources. protocols.io https://dx.doi.org/10.17504/protocols.io.ux8exrw Copy   

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Authors: Elizabeth Smith
Group: SPARC
Summary: Immunohistochemistry protocol used for staining with fluorescent secondary antibodies to highlight specific tissue structures and amplify specific antibody signals.

Proper citation: Elizabeth Smith 2019. IHC-Amplified Fluorescent Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.8rmhv46 Copy   

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Authors: Sean Seaver
Summary: How to make a 20 mg/mL X-Gluc Stock Solution

Proper citation: Sean Seaver 2014. How to make a 20 mg/mL X-Gluc Stock Solution. protocols.io https://dx.doi.org/10.17504/protocols.io.ciyufv Copy   

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Authors: Elizabeth Jannaman, Peter Hansen
Group: University of Florida Bovine Embryo Lab
Summary: A simple protocol to remove the zona pellucida from bovine embryos. This is a necessary step before freezing embryos for analysis of RNA or DNA because of contaminating cumulus and sperm cells associaed with the zona pellucida.

Proper citation: Elizabeth Jannaman, Peter Hansen 2019. Zona pellucida removal using Acid Tyrode solution and snap freezing. protocols.io https://dx.doi.org/10.17504/protocols.io.6kwhcxe Copy   

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Authors: Sarah Giuliani
Summary: Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.As a minimum, prepare 12 sample bottles for cells infected with phage and control phage lysate, under 2 different media treatments, with triplcates of each unique Phage/Treatment combination.

Proper citation: Sarah Giuliani 2016. Setting up Experimental Cultures for Large-scale One-step Phage Infection of Cyanobacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.fktbkwn Copy   

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Authors: Talita Gagliardi
Summary: Total RNA extraction using TRizol reagent.Attention: for nasopharyngeal aspirates, we use regular TRizol reagent in this assay, but for blood samples it is better to use a special one as "TRizol LS".

Proper citation: Talita Gagliardi 2019. Adapted protocol to extract total RNA using TRIzol® (Invitrogen, Thermo). protocols.io https://dx.doi.org/10.17504/protocols.io.w8vfhw6 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the "quick" version of Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030). For the full protocol, please click here.

Proper citation: New England Biolabs 2018. Quick Protocol for Monarch® PCR & DNA Cleanup Kit (5 ?g) (NEB #T1030). protocols.io https://dx.doi.org/10.17504/protocols.io.n3vdgn6 Copy   

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