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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 dx.doi.org/10.17504/protocols.io.e2ibgce Copy
Authors: Kelsey Miller
Group: BioLegend
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 your 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™ Isolation Kits No Wash Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e2wbgfe Copy
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 dx.doi.org/10.17504/protocols.io.hv4b68w Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Th9 Polarization of Mouse CD4+ Cells. protocols.io dx.doi.org/10.17504/protocols.io.e2cbgaw Copy
Authors: Kelsey Miller
Summary: Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol A. If it is higher than 50% then please follow protocol B.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™ Human CD45 Nanobeads Protocol A. protocols.io dx.doi.org/10.17504/protocols.io.e2zbgf6 Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: ACUITYAdvanced Biotin Free HRP
Polymer Detection System for Immunohistochemistry
Proper citation: Kelsey Miller 2016. ACUITYAdvanced Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e37bgrn Copy
Authors: Kelsey Miller
Group: BioLegend
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 your 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™ Isolation Kits Regular Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e2vbge6 Copy
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.Product description and procedure summary: The kit is designed for the isolation of human memory CD4 T cells from PBMC. 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.
Proper citation: Kelsey Miller 2016. MojoSort™ Human CD4 Memory T Cells Isolation Kit Protocol. protocols.io dx.doi.org/10.17504/protocols.io.fxqbpmw 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 5pmol 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 2018. Anti-Neu5Gc Antibody Kit Protocol - ELISA. protocols.io dx.doi.org/10.17504/protocols.io.tigekbw Copy
Authors: Kelsey Miller
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 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.
Proper citation: Kelsey Miller 2016. Anti-Neu5Gc Antibody Kit Protocol - Flow Cytometry. protocols.io dx.doi.org/10.17504/protocols.io.e26bghe Copy
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 2016. Immunofluorescent Staining of Foxp3 in Frozen Sections. protocols.io dx.doi.org/10.17504/protocols.io.e3pbgmn Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: 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 Regular Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e3hbgj6 Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. ELISPOT Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e29bgh6 Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: This protocol has been optimized to remove washing steps after antibody and nanobeads incubation, resulting in a shorter and more convenient protocol. This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. 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.Product description and procedure summary:This kit is designed for the sequential positive selection of CD4+ T cells from human peripheral blood mononuclear cells (PBMCs). Human monocytes express both CD14 and CD4. When using only CD4 Nanobeads for positive selection of human CD4 T cells, monocytes could be isolated along with the T cells. If this monocyte fraction does not impact your application, there is no need to address it. However, not including this population may be required. Thus, the first step in this kit is the depletion of CD14+ cells using a combination of biotin anti-human CD14 and Streptavidin Nanobeads. The second step is the positive selection of the CD4 T cells using directly conjugated CD4 Nanobeads. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Proper citation: Kelsey Miller 2016. MojoSort™ Human CD4 T Cell Selection Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e3gbgjw Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: ACUITYAdvanced Biotin Free HRP
Polymer Detection System for Immunohistochemistry
Proper citation: Kelsey Miller 2017. ACUITYAdvanced Protocol. protocols.io dx.doi.org/10.17504/protocols.io.hvjb64n Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: Anti-BrdU Staining Protocol using DNAse with surface and fluorescent proteins
Proper citation: Kelsey Miller 2016. Anti-BrdU Staining Protocols Using DNAse with Surface and Fluorescent Proteins. protocols.io dx.doi.org/10.17504/protocols.io.e2nbgde Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Immunofluorescence Microscopy Protocol with Methanol Fixed Cells. protocols.io dx.doi.org/10.17504/protocols.io.fkbbksn Copy
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 dx.doi.org/10.17504/protocols.io.e3ibgke Copy
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 2018. Flex-T™ HLA Class I ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.tkaekse Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2018. Immunocytochemistry Staining for Methanol Fixed Cells. protocols.io dx.doi.org/10.17504/protocols.io.tdhei36 Copy
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