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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Sam Li 2019. MojoSort™ Human anti-PE Nanobeads Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7aghibw Copy
Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads and less antibody cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Sam Li 2020. MojoSort™ Human CD14+ Monocyte Isolation Kit Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bbwaipae Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Immunohistochemistry Protocol for Sternberger Monoclonal Antibodies. protocols.io dx.doi.org/10.17504/protocols.io.e2jbgcn Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA). protocols.io dx.doi.org/10.17504/protocols.io.fq9bmz6 Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Intracellular Flow Cytometry Staining Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bac7iazn Copy
Authors: Sam Li
Group: BioLegend
Summary: Anti-Neu5Gc may be used for staining cells prior to analysis by Flow Cytometry. This kit contains all the essential components needed to identify Neu5Gc on the surface of cells by flow cytometry.Use of the blocking agent (Neu5Gc Assay Blocking Solution) provided in the kit is essential, as commonly used blocking agents invariably contain serum, or serum components, that can either inhibit detection or introduce Neu5Gc contamination.Tissue culture-grown CHO-K1 cells can be used as a positive control, and human peripheral blood mononuclear cells serve as a negative control. Adherent tissue culture-grown cells should be released from the culture flasks by using 5-10mM EDTA for 10 minutes at room temperature. Other non-enzymatic methods, such as Accutase®, may be used. Immediately wash cells in blocking buffer that contains a lower concentration of EDTA, and resuspend cells in blocking buffer to determine cell numbers and viability.It is assumed that the user is familiar with the general principles and practices of Flow Cytometry.
Proper citation: Sam Li 2019. Anti-Neu5Gc Antibody Kit Protocol - Flow Cytometry. protocols.io dx.doi.org/10.17504/protocols.io.baaqiadw Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2017. Immunofluorescence Microscopy Protocol with Methanol Fixed Cells. protocols.io dx.doi.org/10.17504/protocols.io.hv9b696 Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Cell Fixation and Permeabilization Protocol using 70% Ethanol. protocols.io dx.doi.org/10.17504/protocols.io.bacziax6 Copy
Authors: BioLegend, Inc.
Group: BioLegend
Summary: This is a collection of BioLegend protocols for the Intracellular Flow Cytometry Staining, including the Activation and Intracellular Staining of Whole Blood.
Proper citation: BioLegend, Inc. 2016. Intracellular Flow Cytometry Staining (Collection). protocols.io dx.doi.org/10.17504/protocols.io.ez4bf8w Copy
Authors: Sam Li
Group: BioLegend
Summary: To view images that accompany this protocol, click here.This protocol provides step-by-step instructions for the setup of Cytek® Aurora and Northern Lights flow cytometers for data acquisition of BioLegend’s LEGENDplex™ multiplex immunoassays. A pre-made LEGENDplex™ Experiment Template that can easily be imported into the SpectroFlo® software is available for download at biolegend.com/en-us/legendplex under the Instrument Setup tab. If desired, users can also create their own Experiment Template by following the instructions found at the end of this protocol. Unmixing of spectral data is not required. Please export Raw FCS files for downstream analysis. This is analogous to using uncompensated data on a conventional flow cytometer. When analyzing LEGENDplex™ Assay FCS files generated on an Aurora or Northern Lights flow cytometer, the assay beads emission spectra should be analyzed using the following recommended channels: • Without a yellow-green (YG) laser: Bead Classification = R4, Reporter = B4 • With a yellow-green (YG) laser: Bead Classification = R4, Reporter = YG1
Proper citation: Sam Li 2020. LEGENDplex™ Assay Setup Protocol for Cytek® Aurora and Northern Lights Flow Cytometers. protocols.io dx.doi.org/10.17504/protocols.io.bi3mkgk6 Copy
Authors: Kelsey Miller, Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads and less antibody cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Kelsey Miller, Sam Li 2019. MojoSort™ Streptavidin Nanobeads Column Protocol - Negative Selection. protocols.io dx.doi.org/10.17504/protocols.io.693hh8n Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Th17 Polarization of Mouse CD4+ Cells. protocols.io dx.doi.org/10.17504/protocols.io.79vhr66 Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2018. Immunohistochemistry Protocol for Keratin Antibodies. protocols.io dx.doi.org/10.17504/protocols.io.tkhekt6 Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Cell Surface Flow Cytometry Staining Protocol. protocols.io dx.doi.org/10.17504/protocols.io.baa9iah6 Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Propidium Iodide Cell Cycle Staining Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e2mbgc6 Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: This protocol is essentially the same as a standard ELISA protocol, with one major exception – all animal compounds must be avoided in buffers, etc. because they contain Neu5Gc, which will interfere with antibody activity and result in misleading data. Essentially, the glycoconjugate to be evaluated is immobilized on an ELISA plate and exposed to a primary antibody. The antibody provided in this kit has been shown to identify as little as 5 pmol of Neu5Gc per microgram glycoprotein, which is at or below the current detection limit for conventional DMB HPLC analysis. An advantage of the ELISA procedure is that it confirms the presence of Neu5Gc on the glycoconjugate of interest, but use caution, as it also reacts with solution/media that contains glycoconjugate.
Proper citation: Kelsey Miller 2017. Anti-Neu5Gc Antibody Kit Protocol - ELISA. protocols.io dx.doi.org/10.17504/protocols.io.hvpb65n Copy
Authors: BioLegend, Inc.
Group: BioLegend
Proper citation: BioLegend, Inc. 2016. Sandwich ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.ex9bfr6 Copy
Authors: Vinodkumar Kulangara, Neetha Nair, Asok Sivasailam, Suchithra Sasidharan, Justin Davis and Radhika Syam
Group: BioLegend
Summary: Dairy cows affected with subclinical mastitis can be sources of virulent, antimicrobial-resistant Staphylococci to humans by the excretion of the bacteria through their milk. This study focussed on the phenotypic and genotypic antibiotic resistance patterns of Staphylococci isolated from dairy cows during the early dry period. Among 96 isolates of Gram positive cocci from 157 cows, 76 were identified as Coagulase Negative Staphylococci and the remaining twenty were Staphylococcus aureus. Typical amplicons of coagulase gene were obtained for all twenty samples of S. aureus with three major coagulase types being identified as giving 627 bp (40 %), 910 bp (35 %) and 710 bp (25 %) long PCR products. The groEL gene was amplified in PCR of all 76 isolates of Coagulase Negative Staphylococci, and incubation of PCR products with restriction enzyme PvuII yielded three distinct PCR-RFLP fragment patterns bearing resemblance to S. chromogenes and S. hyicus. Highest sensitivity of Coagulase Negative Staphylococci was noted for Azithromycin (92.5%) and the least to Tetracyclines (76.3%), whereas for S. aureus, it was Cefoperazone (95%) and Azithromycin (72.2%) respectively. Phenotypic resistance to Oxacillin (25 isolates), and Cefoxitin (11 isolates) was detected by dilution method with a commercial strip (Ezy MICTM). Genotypic resistance to b-Lactam antibiotics was found in 65 (34 with mecA gene and 31 with blaZ gene) isolates. Eighteen isolates possessed both the genes, with the PVL gene for virulence being detected in five of them. Nine isolates which had mecA gene were phenotypically susceptible to oxacillin while phenotypic resistance to oxacillin was observed in seven isolates that did not have either mecA or blaZ gene. This is the first report of persistent Staphylococcal infections possessing PVL gene and high level of genotypic resistance to b-Lactam antibiotics in small- holder dairy cattle from India.
Proper citation: Vinodkumar Kulangara, Neetha Nair, Asok Sivasailam, Suchithra Sasidharan, Justin Davis and Radhika Syam 2017. Genotypic and phenotypic ?-Lactam resistance and presence of PVL gene in Staphylococci from dry bovine udder . protocols.io dx.doi.org/10.17504/protocols.io.jhccj2w Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: Anti-BrdU Staining Protocol Using 70% Ethanol and 2N HCl
Proper citation: Kelsey Miller 2017. Anti-BrdU Staining Using 70% Ethanol and 2N HCl. protocols.io dx.doi.org/10.17504/protocols.io.hvmb646 Copy
Authors: Sam Li
Group: BioLegend
Summary: Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol 1. If it is higher than 50% then please follow protocol 2.The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Proper citation: Sam Li 2019. MojoSort™ Human CD45 Nanobeads Protocols. protocols.io dx.doi.org/10.17504/protocols.io.7wrhpd6 Copy
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