Anti cd3 pb

Manufactured by BioLegend

Sourced in United States

Anti-CD3-PB is a fluorochrome-conjugated antibody that binds to the CD3 antigen on the surface of T cells. It is a commonly used tool in flow cytometry applications for the identification and enumeration of T cell populations.

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Anti-CD3 (468 citations) Anti-human CD3 PB (2 citations)

3 protocols using anti cd3 pb

Multiparameter Flow Cytometric Phenotyping

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D10-19 M1₅₈ cultures were stained with HLA-A*02:01-M1₅₈ tetramer conjugated to PE or APC at a 1:100 dilution in FACS buffer (PBS with 0.1% bovine serum albumin) or PBS. Cells were then washed twice with cold FACS buffer and stained with a cocktail of antibodies including anti-CD3-PB (Biolegend) or anti-CD3-PeCy7 (eBiosciences), anti-CD8-PerCPCy5.5 or anti-CD8-PerCP (both BD Biosciences), CD27-APC or APC-Cy7 (BD Biosciences), CD45RA-FITC (BD Pharmingen, San Diego, CA, USA) and Live/Dear-NIR (Invitrogen) (Indigenous donors only), washed twice with FACS buffer or PBS. Cells were then resuspended in 200 μl of sort buffer and passed though a 40-μm sieve prior to flow cytometric analysis or sorting.

Clemens E.B., Grant E.J., Wang Z., Gras S., Tipping P., Rossjohn J., Miller A., Tong S.Y, & Kedzierska K. (2015). Towards identification of immune and genetic correlates of severe influenza disease in Indigenous Australians. Immunology and Cell Biology, 94(4), 367-377.

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Monocyte-derived Dendritic Cell Activation

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PBMC were thawed and monocytes were isolated by positive selection using MACS CD14 MicroBeads (MiltenyiBiotec, Auburn, CA, USA) following manufacturer’s instructions. Monocytes were seeded in T75 flasks (Corning) in RPMI medium containing 10% heat-inactivated fetal calf serum (FCS, 50 U/ml penicillin, 50 µg/ml streptomycin (Gibco, Paisley, United Kingdom), IL-4 and GM-CSF (Biosource International, Camarillo, CA, USA) both at 10 ng/ml for 6 days to generate monocyte-derived immature DCs. Mature DCs were generated by stimulating DCs with 10 ng/ml lipopolysaccharide (LPS) for an additional 24 hours. DC differentiation and maturation was validated by flow cytometry using anti-CD14 PE (BD Pharmingen), anti-HLA-DR FITC (BD Pharmingen), anti-CD1a Alexa Fluor 700 (Biolegend), anti-CD163 Alexa Fluor 647 (Biolegend), anti-CD80 PE-Cy7 (Biolegend), anti-CD86 PE-Cy5 (BD Pharmingen) and anti-CD3 PB (Biolegend).
Matured DCs were loaded with Rv2034 protein, Rv2034 p81–100 and different conditions of Mtb lysate and incubated for 24 hours. Cells were washed and T-cell clone added. After 2 hours BFA was added and culture incubated o/n. Activation of T cells was determined by detection of CD154 and Th1 markers using the T-helper subset panel.

Commandeur S., Coppola M., Dijkman K., Friggen A.H., van Meijgaarden K.E., van den Eeden S.J., Wilson L., van der Ploeg-van Schip J.J., Franken K.L., Geluk A, & Ottenhoff T.H. (2014). Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method. PLoS ONE, 9(6), e99203.

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Multiparametric Flow Cytometry of Tumor Immune Cells

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Single cell suspensions from dissociated tumors were washed with PBS containing 1% FBS and stained with the following antibody cocktails. Lymphoid antibody cocktail contained: anti-CD45-PerCP/Cy5.5 (BioLegend 103132), anti-NK1.1-FITC (BioLegend 108706), anti-CD3-PB (BioLegend 100214), anti-CD4-APC (BioLegend 100412), anti-CD8-AF700 (BioLegend 100730), and anti-PD-1(CD279)-PE/Cy7 (BioLegend 135216). Myeloid antibody cocktail: anti-CD45-PerCP/Cy5.5 (BioLegend 103132), anti-CD19-FITC (BioLegend 115506), anti-B220-FITC (BioLegend 103206), anti-CD3-FITC (BioLegend 100204), anti-CD11b(Mac1)-PB (BioLegend 101224), anti-CD11c-PE/Cy7 (BioLegend 117318), and anti-Ly-6G(Gr1)-AF700 (BioLegend 127622). Propidium Iodide was used to detect dead cells. Samples were run on an Attune Nxt Flow Cytometer (ThermoFisher Scientific) at the Siteman Flow Cytometry Core Facility. Analysis was done in FlowJo V10 and statistically significant differences were identified using One-way ANOVA.

Zimmerman S.M., Nixon S.J., Chen P.Y., Raj L., Smith S.R., Paolini R.L., Nay Lin P, & Souroullas G.P. (2022). Ezh2Y641F mutations co-operate with Stat3 to regulate MHC Class I antigen processing and alter the tumor immune response in melanoma. Oncogene, 41(46), 4983-4993.

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