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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 2017. Chromatin Immunoprecipitation (ChIP) Assay Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.humb6u6 Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: The cells targeted by the Nanobeads are 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 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Mouse CD45 Nanobeads Protocol - Depletion. protocols.io https://dx.doi.org/10.17504/protocols.io.7wkhpcw Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2016. Sandwich ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ex9bfr6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.7wrhpd6 Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Th9 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.e2cbgaw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.hvmb646 Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2016. Mouse T Cell Activation with anti-CD3? Antibodies (clone 145-2C11). protocols.io https://dx.doi.org/10.17504/protocols.io.exubfnw 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 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™ Mouse anti-PE Nanobeads Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7achiaw 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 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 2019. MojoSort™ Mouse NK Cell Isolation Kit Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7bhhij6 Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. LEGEND MAX™ Human α-Synuclein ELISA Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.98th9wn Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: Product description and procedure summary:Target cells are either selected or depleted by incubating your sample with the biotin anti-human CD14 antibody (clone 63D3) followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the CD14+ cells, do not discard them if those are your cells of interest. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.

Proper citation: Kelsey Miller 2016. MojoSort™ Human CD14 Selection Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e3jbgkn Copy   

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Authors: Kelsey Miller
Group: BioLegend

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

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Authors: Sam Li
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: Sam Li 2019. Flex-T™ HLA Class I ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.98wh9xe Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: Anti-BrdU Staining Protocol Using 70% Ethanol and 2N HCl

Proper citation: Kelsey Miller 2018. Anti-BrdU Staining Using 70% Ethanol and 2N HCl. protocols.io https://dx.doi.org/10.17504/protocols.io.tibekan Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: T regulatory cells (also known as Tregs or Regulatory T cells) are essential cells in the immune system that suppress immune responses of other cells, designed to limit excessive reactions and prevent autoimmunity. Tregs are characterized by the expression of CD4, CD25, and Foxp3, while lacking CD127. CD4+Foxp3+ regulatory T cells have been referred to as “naturally-occurring” regulatory T cells to distinguish them from “suppressor” T cell populations that are generated in vitro. While other variants of suppressive T cells do exist, such as CD8 suppressor cells, Th3 and Tr-1 cells, Tregs are classically defined as CD4+CD25+Foxp3+ cells. Here, we provide a protocol for immunofluorescent staining of FOXP3 in frozen sections, adapted from a protocol from Dr. Matthias Hardtke-Wolenski, Medizinische Hochschule Hannover. This protocol has been successfully used on mouse spleen, liver, and skin sections.

Proper citation: Kelsey Miller 2017. Immunofluorescent Staining of Foxp3 in Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.hwbb7an Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Preparation of Brain Samples for LEGEND MAX™ Beta Amyloid ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.98vh9w6 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 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 2019. MojoSort™ Isolation Kits Column Protocol - 4. protocols.io https://dx.doi.org/10.17504/protocols.io.7bfhijn Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are depleted by incubating your 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. These are the cells of interest; do not discard the liquid. 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 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Isolation Kits Protocol - 4. protocols.io https://dx.doi.org/10.17504/protocols.io.7x8hprw Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: Anti-BrdU Staining Protocol Using 70% Ethanol and 2N HCl

Proper citation: Kelsey Miller 2016. Anti-BrdU Staining Using 70% Ethanol and 2N HCl. protocols.io https://dx.doi.org/10.17504/protocols.io.e2pbgdn Copy   

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Authors: BioLegend, Inc.
Group: BioLegend
Summary: This is a collection of Western Blotting protocols from BioLegend. Please use the appropriate protocol, depending on your application.

Proper citation: BioLegend, Inc. 2016. Western Blotting Protocols (Collection). protocols.io https://dx.doi.org/10.17504/protocols.io.e2bbgan Copy   

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