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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: If your target cells are the labeled cells (the positive fraction), use the Streptavidin Nanobeads Protocol – Positive Selection. If your target cells are the unlabeled cells (negative fraction), use the Streptavidin Nanobeads Protocol - Negative Selection. Target cells are isolated by incubating your sample with a Biotin-conjugated antibody or 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 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Streptavidin Nanobeads Protocol - Negative Selection. protocols.io https://dx.doi.org/10.17504/protocols.io.7u8hnzw Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: This protocol has been optimized to remove washing steps after antibody cocktail and nanobeads incubations, resulting in a shorter and more convenient protocol. This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube from human peripheral blood mononuclear cells (PBMCs). If working with fewer 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. To maximize the purity of CD4+ T cells we recommend the depletion of monocytes, as human monocytes express CD4. To deplete monocytes from human PBMCs, we recommend the use of MojoSort™ Human CD14 Selection Kit (Cat. No. 480025/480026). Alternatively, you may consider the Human CD4 T Cell Selection Kit (Cat. No. 480038). After monocyte depletion proceed to isolate the CD4+ T cells. You can also use other methods to deplete monocytes. If you wish to include monocytes in your CD4+ cell isolation, depletion is not required

Proper citation: Kelsey Miller 2016. MojoSort™ Human CD4 Nanobeads No Wash Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e3ibgke Copy   

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Authors: Kelsey Knight
Group: BioLegend
Summary: 1. Activated cell populations can be prepared from in vivo-stimulated tissues or from in vitro-stimulated cultures (e.g., antigen-specific activation or mitogen-induced). For cytokine and chemokine detection, it is critical to include a protein transport inhibitor such as brefeldin A (BioLegend Cat. No.420601) or monensin (BioLegend Cat. No.420701) in the last 4-6 hours of cell culture activation. The cells can be suspended and distributed to 12 x 75 mm plastic tubes or microwell plates for immunofluorescent staining. For details on stimulation methods, please see our stimulation guide for cytokines/chemokines: (www.biolegend.com/media_assets/support_protocol/BioLegend_StimulationGuide_101711.pdf)2. Different cytokines/chemokines have different production peaks. In order to obtain optimal staining signals, the stimulation conditions for each stimulant need to be optimized.3. Some antibodies recognizing native cell surface markers may not bind to fixed/denatured antigens. For this reason, it is recommended that staining of cell surface antigens be done with live, unfixed cells PRIOR to fixation/permeabilization and staining of intracellular targets. Altering the procedure such that cells are fixed prior to staining of cell surface antigens requires that paraformaldehyde-denatured antigen reactive antibody clones be empirically identified. You can also take a look at our Fixation Webpage (www.biolegend.com/fixation) to get an idea of how epitopes stain after fixation with 4% PFA. Please note that the ability to stain post-fixation depends on the fluor, antigen expression, and several other factors.

Proper citation: Kelsey Knight 2018. Intracellular Flow Cytometry Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tknekve Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Immunofluorescence Microscopy Protocol with Methanol Fixed Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.fkbbksn Copy   

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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.The following protocol combines highly multiplexed protein marker detection with unbiased transcriptome profiling for thousands of single cells. Epitope detection and sample multiplexing (Cell Hashing) are enabled by BioLegend’s TotalSeq™ antibody-oligo conjugates. TotalSeq™ reagents are compatible with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), RNA Expression And Protein sequencing assay (REAP-seq), and similar workflows.

Proper citation: Sam Li 2019. TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v2. protocols.io https://dx.doi.org/10.17504/protocols.io.8v3hw8n Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Mouse CD326 (Ep-CAM) cells are selected by incubating the sample with biotin conjugated anti-mouse CD326 antibody after blocking with TruStain FcX™ (anti-mouse CD16/32 antibody) then followed by Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the CD326 (Ep-CAM) expressing cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc. In addition to CD326 (Ep-CAM) expression on epithelial and tumor cells, CD326 is also expressed on subtypes of leukocytes, which should be depleted using MojoSort™ Mouse CD45 Nanobeads (Cat. No. 480027/480028) before CD326 positive selection. 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 CD326 (Ep-CAM) Positive Selection Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.72ahqae Copy   

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Authors: BioLegend, Inc.
Group: BioLegend
Summary: This is a collection of BioLegend protocols for Immunofluorescence Staining.

Proper citation: BioLegend, Inc. 2016. Immunofluorescence Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.ezvbf66 Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Anti-DYKDDDDK Tag (L5) Affinity Gel Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.98kh9uw Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Th2 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.796hr9e Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Astrocytes are the major glial cells in the central nervous system and are involved in the regulation of numerous processes like energy metabolism, ion homeostasis, and synaptic plasticity. Primary astrocytes derived from rodent brains provide a rapid and easy solution for studying astrocyte biology and morphology. These cultured cells are an invaluable tool for elucidating underlying mechanisms of diseases such as: 1Astrocytes are the major glial cells in the central nervous system and are involved in the regulation of numerous processes like energy metabolism, ion homeostasis, and synaptic plasticity. Primary astrocytes derived from rodent brains provide a rapid and easy solution for studying astrocyte biology and morphology. These cultured cells are an invaluable tool for elucidating underlying mechanisms of diseases such as:StrokeEpilepsyAmyotrophic lateral sclerosisParkinson’s diseaseAlzheimer’s diseaseTraumaThis protocol demonstrates how to isolate and culture primary astrocytes from mouse pup brains. The purity and morphology of astrocytes is assessed by immunocytochemistry (ICC) using well-established astrocytic markers, including GFAP.StrokeEpilepsyAmyotrophic lateral sclerosisParkinson’s diseaseTraumaAlzheimer's disease 1Astrocytes are the major glial cells in the central nervous system and are involved in the regulation of numerous processes like energy metabolism, ion homeostasis, and synaptic plasticity. Primary astrocytes derived from rodent brains provide a rapid and easy solution for studying astrocyte biology and morphology. These cultured cells are an invaluable tool for elucidating underlying mechanisms of diseases such as:StrokeEpilepsyAmyotrophic lateral sclerosisParkinson’s diseaseAlzheimer’s diseaseTraumaThis protocol demonstrates how to isolate and culture primary astrocytes from mouse pup brains. The purity and morphology of astrocytes is assessed by immunocytochemistry (ICC) using well-established astrocytic markers, including GFAP. 1Astrocytes are the major glial cells in the central nervous system and are involved in the regulation of numerous processes like energy metabolism, ion homeostasis, and synaptic plasticity. Primary astrocytes derived from rodent brains provide a rapid and easy solution for studying astrocyte biology and morphology. These cultured cells are an invaluable tool for elucidating underlying mechanisms of diseases such as:StrokeEpilepsyAmyotrophic lateral sclerosisParkinson’s diseaseAlzheimer’s diseaseTraumaThis protocol demonstrates how to isolate and culture primary astrocytes from mouse pup brains. The purity and morphology of astrocytes is assessed by immunocytochemistry (ICC) using well-established astrocytic markers, including GFAP. 1Astrocytes are the major glial cells in the central nervous system and are involved in the regulation of numerous processes like energy metabolism, ion homeostasis, and synaptic plasticity. Primary astrocytes derived from rodent brains provide a rapid and easy solution for studying astrocyte biology and morphology. These cultured cells are an invaluable tool for elucidating underlying mechanisms of diseases such as:StrokeEpilepsyAmyotrophic lateral sclerosisParkinson’s diseaseAlzheimer’s diseaseTraumaThis protocol demonstrates how to isolate and culture primary astrocytes from mouse pup brains. The purity and morphology of astrocytes is assessed by immunocytochemistry (ICC) using well-established astrocytic markers, including GFAP. 1Astrocytes are the major glial cells in the central nervous system and are involved in the regulation of numerous processes like energy metabolism, ion homeostasis, and synaptic plasticity. Primary astrocytes derived from rodent brains provide a rapid and easy solution for studying astrocyte biology and morphology. These cultured cells are an invaluable tool for elucidating underlying mechanisms of diseases such as:StrokeEpilepsyAmyotrophic lateral sclerosisParkinson’s diseaseAlzheimer’s diseaseTraumaThis protocol demonstrates how to isolate and culture primary astrocytes from mouse pup brains. The purity and morphology of astrocytes is assessed by immunocytochemistry (ICC) using well-established astrocytic markers, including GFAP. 1Astrocytes are the major glial cells in the central nervous system and are involved in the regulation of numerous processes like energy metabolism, ion homeostasis, and synaptic plasticity. Primary astrocytes derived from rodent brains provide a rapid and easy solution for studying astrocyte biology and morphology. These cultured cells are an invaluable tool for elucidating underlying mechanisms of diseases such as:StrokeEpilepsyAmyotrophic lateral sclerosisParkinson’s diseaseAlzheimer’s diseaseTraumaThis protocol demonstrates how to isolate and culture primary astrocytes from mouse pup brains. The purity and morphology of astrocytes is assessed by immunocytochemistry (ICC) using well-established astrocytic markers, including GFAP.

Proper citation: Sam Li 2019. Isolation and Culture of Primary Mouse Astrocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.97eh9je Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Ki-67 Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bac2iaye Copy   

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Authors: Kelsey Miller
Group: BioLegend
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.

Proper citation: Kelsey Miller 2016. MojoSort™ Nanobeads No Wash Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e2tbgen Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: 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. The purity of the CD3- CD49b+ NK Cell fraction typically ranges from 80 to 90%. Removal of dead cells is highly recommended prior to cell isolation. Dead cells can be removed using appropriate centrifugation media or other methods.Product description and procedure summary: The kit is designed for the isolation of mouse NK cells from lymphoid tissues. Target cells are depleted by incubating the 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.

Proper citation: Kelsey Miller 2016. MojoSort™ Mouse NK Cell Isolation Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.fxpbpmn Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2016. Th2 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.ezqbf5w Copy   

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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 2016. Anti-Neu5Gc Antibody Kit Protocol - ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.e27bghn Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA). protocols.io https://dx.doi.org/10.17504/protocols.io.95rh856 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 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   

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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   

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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   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Sandwich ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tm9ek96 Copy   

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