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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 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™ Selection Kits Column Protocol - 3. protocols.io https://dx.doi.org/10.17504/protocols.io.7tvhnn6 Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: This kit is designed for the isolation of untouched CD14+CD16- monocytes from peripheral blood mononuclear cells (PBMCs). Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. 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™ Human CD14+ Monocytes Isolation Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7x9hpr6 Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Propidium Iodide Cell Cycle Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bacyiaxw Copy
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 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.
Proper citation: Sam Li 2019. MojoSort™ Mouse CD4 Nanobeads Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7wthpen Copy
Authors: Sam Li
Group: BioLegend
Summary: Catalog Number: 658502Storage Temperature: 2°C-8°CProduct Description: Anti-c-Myc Tag (9E10) Affinity Gel consists of anti-c-myc monoclonal antibody (clone 9E10), covalently immobilized onto 6% high density glyoxal agarose beads. The affinity resin can be used in affinity purifying and immunoprecipitation of c-Myc-tagged fusion protein.Binding Specificity: Mouse monoclonal antibody 9E10 recognizes the c-myc epitope N-EQKLSEEDL-C and is purified through Protein G chromatography. The antibody conjugated affinity resin can bind to epitope at N-terminal, C-terminal, and internal locations of a fusion protein. The binding capacity is greater than 0.5mg per ml of c-myc agarose resin.Reagent: Anti-c-Myc Agarose Affinity Gel is supplied as a 50% suspension in phosphate-buffered saline, 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-c-Myc Agarose Affinity Gel should be stored between 2°C and 8°C for maximum stability. The unopened product is stable for one year when stored as indicated. After use, the resin should be regenerated and stored in TBS or PBS buffer (pH 7.2) containing 0.09% sodium azide.
Proper citation: Sam Li 2019. Anti-c-Myc Tag (9E10) Affinity Gel Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.97ih9ke 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 in 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 2020. MojoSort™ Mouse Ig light chain κ Nanobeads Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bbueinte Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Cell Surface Flow Cytometry Staining of Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.babbiain 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 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™ Selection Kits Column Protocol - 5. protocols.io https://dx.doi.org/10.17504/protocols.io.7u3hnyn Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. True-Nuclear™ Transcription Factor Staining Protocol for 5 mL Tubes. protocols.io https://dx.doi.org/10.17504/protocols.io.bacsiawe Copy
Authors: Sam Li
Group: BioLegend
Summary: Using UV-induced peptide exchange, MHC/peptide monomers can be generated with conditional Flex-T™ monomers that harbor peptides of interest in their binding grooves. These new MHC monomers are subsequently multimerized using streptavidin-fluorophore conjugates. The resulting Flex-T™ reagents can be used for staining antigen-specific T cells and flow cytometric analysis. In humans, the MHC molecules are called HLA (Human Leukocyte Antigen).
Proper citation: Sam Li 2019. Flex-T™ Tetramer and Cell Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.babeiaje Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Cell Surface Flow Cytometry Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.baa9iah6 Copy
Authors: Sam Li
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: Sam Li 2019. Immunofluorescent Staining of Foxp3 in Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.97ch9iw 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 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 https://dx.doi.org/10.17504/protocols.io.7aghibw 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 https://dx.doi.org/10.17504/protocols.io.baaqiadw 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 https://dx.doi.org/10.17504/protocols.io.bbwaipae Copy
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 APC conjugated antibody, followed by incubation with magnetic anti-APC Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the APC+ 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-APC Nanobeads Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7x2hpqe Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Cell Fixation and Permeabilization Protocol using 70% Ethanol. protocols.io https://dx.doi.org/10.17504/protocols.io.bacziax6 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 https://dx.doi.org/10.17504/protocols.io.693hh8n Copy
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.
Proper citation: Sam Li 2019. MojoSort™ Nanobeads Protocol - 1. protocols.io https://dx.doi.org/10.17504/protocols.io.7u5hny6 Copy
Authors: Sam Li
Group: BioLegend
Summary: Buyer is solely responsible for determining whether Buyer has all intellectual property rights that are necessary for Buyer's intended uses of the BioLegend TotalSeq™ products. For example, for any technology platform Buyer uses with TotalSeq™, it is Buyer's sole responsibility to determine whether it has all necessary third party intellectual property rights to use that platform and TotalSeq™ with that platform.
TotalSeq™-B antibodies are designed to be used with 10x Single Cell 3’ Reagent Kit v3 with Feature Barcoding. The adjusted protocol below is for customers who are utilizing TotalSeq™-A antibodies with the v3 kit instead. Note: Step 4 from the 10x Genomics user guide document number CG000185, Rev B only applies to the cDNA library and NOT to the TotalSeq™-A library.
Please read the entire protocol below and the 10x Genomics user guide for the Chromium Single Cell 3ʹ Reagent Kits v3 with Feature Barcoding technology for Cell Surface Protein before starting the experiments. 10x Genomics user guide document number CG000185, Rev B.
Commonly used abbreviations:ADT: Antibody derived tags, the oligo sequence conjugated to regular TotalSeq™-A antibodiesHTO: Hashtag oligonucleotides, the oligo sequence conjugated to TotalSeq™-A Hashtag antibodies
Proper citation: Sam Li 2019. TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.8aahsae Copy
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