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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Nuclear Factor Fixation and Permeabilization Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e3bbgin Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Cell Surface Immunofluorescent Staining of Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.tjwekpe Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: Target cells are positively selected or depleted by incubating the sample with an anti-human PE conjugated antibody, followed by incubation with magnetic anti-PE Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the PE+ cells, do not discard them if those are the cells of interest. 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 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Human anti-PE Nanobeads Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7x3hpqn Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: The HLA class I ELISA is an enzyme immunoassay based on the detection of β2-microglobulin subunit of HLA class I complexes, after capturing the complex through the conjugated biotin. To this end, biotinylated HLA class I complex is first captured in streptavidin coated microtiter wells. Subsequently, HRP-conjugated anti-human β2-microglobulin is added to detect intact HLA class I complexes. Only intact HLA class I complexes are recognized. Peptides with high affinity binding will be clearly detected by this ELISA technique, while peptides with a moderate to low binding affinity for HLA class I provide a moderate to non-detectable signal. This protocol is designed to evaluate the efficiency of peptide exchange when using the Flex-T™ system.

Proper citation: Kelsey Miller 2017. Flex-T™ HLA Class I ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.hv4b68w Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Intracellular Flow Cytometry Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bac7iazn Copy   

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Authors: BioLegend, Inc.
Group: BioLegend
Summary: The following is a general procedure guide for preparation and staining of formalin-fixed, paraffin-embedded tissues using a purified, unconjugated primary antibody, biotinylated secondary antibody and streptavidin-horseradish peroxidase (Sav-HRP) and DAB detection system. Because each antigen differs in terms of requirement for fixation, amplification step, etc., it is not possible to write an inclusive protocol that will work for all antigens. The user must determine optimal conditions for each antigen of interest. Many protocols for staining individual antigens, as well as useful tips and troubleshooting guides for immunohistochemistry, can be found at the IHC World web site (http://www.ihcworld.com/).

Proper citation: BioLegend, Inc. 2016. Immunohistochemistry Protocol for Paraffin-Embedded Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.eyxbfxn Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA). protocols.io https://dx.doi.org/10.17504/protocols.io.fq9bmz6 Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Immunohistochemistry Protocol for Beta Amyloid Products using USA Detection Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.e2ibgce Copy   

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Authors: Kelsey Knight
Group: BioLegend
Summary: Catalog Number: 651501, 651502, 651503  Storage Temperature: 4°C Product Description: Anti-DYKDDDDK tag (L5) affinity gel is a purified rat IgG2a, κ monoclonal antibody covalently attached to agarose by hydrazide linkage. It is useful for purification or immunoprecipitation of DYKDDDDK-tagged fusion proteins. The binding of antibody to the DYKDDDDK tag is not calcium dependent. Binding Specificity: Octapeptide (N-DYKDDDDK-C) at N-terminal, Met-N-terminal, C-terminal, and internal locations of a fusion protein. The binding capacity is greater than 0.8mg/ml of L5 agarose resin. Reagent: Anti-DYKDDDDK tag (L5) affinity gel is supplied as a 50% suspension in phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.  Precautions and Disclaimer: This product is for research use only. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.  Storage/Stability: Anti-DYKDDDDK tag (L5) affinity gel should be stored in at 4°C for maximum stability. The unopened product is stable for one year when stored as indicated. After use, the resin should be cleaned and stored in TBS or PBS buffer (pH 7.2) containing 0.09% sodium azide.

Proper citation: Kelsey Knight 2018. Anti-DYKDDDDK Tag (L5) Affinity Gel Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tieekbe Copy   

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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 https://dx.doi.org/10.17504/protocols.io.ez4bf8w Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Reagents - Basic cell culture media: respective to the cell line used and experimental design Distilled water: Invitrogen® UltraPure™ Distilled Water (catalog number 19977015) or equivalent product from other vendor.Highthroughput (HT) Pro A or G 96 well plate or Spin Column (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)96-well collection plate: USA scientific® TempPlate non-skirted 96-well PCR plate natural (catalog number 1402-9596) or equivalent product from other vendor.QIAquick™ PCR purification kit (Qiagen®; catalog number 28104) or equivalent product from other vendor.Hypotonic Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Digestion Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Lysis Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Column Conditioning Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162  or 500163)Wash Buffer 1 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Wash Buffer 2 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Wash Buffer 3 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Elution Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)5M NaCl: dissolve 292g of NaCl in 800mL of H2O. Adjust the volume to 1L with H2O.1M NaHCO3: dissolve 12.6g of NaHCO3 in 100 mL of H2O. Adjust the volume to 150 mL with H2O.Protease Inhibitor Cocktail (PIC): Cell signaling Technology® (catalog number 5871) or equivalent product from other vendor.Proteinase K: Qiagen® (catalog number 19133) or equivalent product from other vendor.Proteinase K stop solution: 100mM Phenylmethylsulfonyl fluoride (PMSF). Weigh 1.742g PMSF (Sigma-Aldrich®, catalog number 10837091001) and add DMSO (Sigma-Aldrich®, catalog number D2650) to a final volume of 100mL. Dissolve completely.PBS: HyClone™ Phosphate Buffered Saline (1X) (catalog number SH30256.01) or equivalent product from other vendor.Formaldehyde: VWR® 37% w/w aqueous solution (catalog number 97064-604) or equivalent product from other vendor.0.65M Glycine: Dissolve 488g of glycine into 1L of H2O to make 6.5M glycine (10X). Dilute with PBS to make 1X before each experiment.   Micrococcal Nuclease: Cell Signaling Technologies® (catalog number 10011S) or equivalent product from other vendor.1.3-1.5% Agarose gel: dissolve 1.3-1.5g of agarose powder into 100mL of TAE buffer by microwave heating. Visualize DNA under UV light by adding appropriate amount of Ethidium Bromide, or use an equivalent method.Add appropriate amount of Ethidium Bromide to cooled down gel solution to visualize DNA under UV light.0.5M EDTA: Dissolve 186.1g EDTA into 700mL of H2O. Adjust the volume to 1L with H2O.100bp ladder: NEB® (catalog number N0467S) or equivalent product from other vendor.Materials -Heat blockRotatorCentrifugeMicrocentrifuge37ºC incubatorNanodropSonicatorUV light imagerRocking platform

Proper citation: Sam Li 2016. Chromatin Immunoprecipitation (ChIP) Assay Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.gsmbwc6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bi3mkgk6 Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Th17 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.79vhr66 Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2017. Immunofluorescence Microscopy Protocol with Methanol Fixed Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.hv9b696 Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: 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.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 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.Application notes: To use this product in magnetic separation columns, a titration of the Nanobeads should be performed. Optimal concentration for magnetic separation columns is lot-specific. Please contact BioLegend Technical Service ([email protected]) for further assistance on how to use MojoSort™ Nanobeads in magnetic separation columns.

Proper citation: Sam Li 2019. MojoSort™ Human CD41 Nanobeads Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7xxhppn Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: The HLA class I ELISA is an enzyme immunoassay based on the detection of β2-microglobulin subunit of HLA class I complexes, after capturing the complex through the conjugated biotin. To this end, biotinylated HLA class I complex is first captured in streptavidin coated microtiter wells. Subsequently, HRP-conjugated anti-human β2-microglobulin is added to detect intact HLA class I complexes. Only intact HLA class I complexes are recognized. Peptides with high affinity binding will be clearly detected by this ELISA technique, while peptides with a moderate to low binding affinity for HLA class I provide a moderate to non-detectable signal. This protocol is designed to evaluate the efficiency of peptide exchange when using the Flex-T™ system.

Proper citation: Kelsey Miller 2016. Flex-T™ HLA Class I ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e7ebhje Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2016. Th1 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.eznbf5e Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2018. Immunohistochemistry Protocol for Keratin Antibodies. protocols.io https://dx.doi.org/10.17504/protocols.io.tkhekt6 Copy   

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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 than 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 CD4 T Cell Selection Kit Column Protocol - CD14 Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.7txhnpn Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: Cell Fixation and Permeabilization Protocol Using 70% Ethanol

Proper citation: Kelsey Miller 2017. Cell Fixation and Permeabilization Protocol using 70% Ethanol. protocols.io https://dx.doi.org/10.17504/protocols.io.hv3b68n Copy   

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