For each staining, 1 x 106 J-Lat cells were washed once with PBS (Gibco), resuspended in 100 μl PBS, and stained with Zombie dye (Cat. #423105, BioLegend) according to manufacturer’s protocol (1:100 dilution) to enable subsequent discrimination between live and dead cells. 10 min after incubation with the Zombie dye, human CD45-PE (clone HI30, Cat. #304039, BioLegend), CD4-PE (clone OKT4, Cat. #317410, BioLegend), TCR α/β-PE (clone IP26, Cat. #306708, BioLegend), CD27-PE (clone M-T271, Cat. #356406, BioLegend), CD147-APC (clone HIM6, Cat. #306214, BioLegend), CD29-APC (clone TS2/16, Cat. #303008, BioLegend), and HLA-ABC-APC/Cy7 (clone W6/32, Cat. #311426, BioLegend) antibodies were added respectively, and were incubated for another 20-30 min at room temperature (RT) in the dark. Cells were then washed with 2 ml of cell staining buffer (Cat. #420201, BioLegend), resuspended in 300 μl PBS and measured using an LSR II flow cytometer (BD Biosciences). Respective Isotype stained samples were used as control. At least 10,000 events were recorded for each sample.
Cd27 pe clone m t271
Manufactured by BioLegend
CD27-PE (clone M-T271) is a fluorescently labeled monoclonal antibody that binds to the CD27 antigen. CD27 is a member of the tumor necrosis factor receptor superfamily and is expressed on the surface of T cells, B cells, and natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Cell staining buffer, by BioLegend (1 mentions)
Cd4 pe clone okt4, by BioLegend (1 mentions)
Cd29 apc clone ts2 16, by BioLegend (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5.5 clone okt4, by BioLegend (1 mentions)
Cd45ro apc clone uchl1, by BioLegend (1 mentions)
Facsaria 2 cell sorter, by BD (1 mentions)
2 protocols using cd27 pe clone m t271
1
Multiparametric flow cytometry analysis
2
CD4+ T Cell Subset Analysis and Sorting
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CD4 subset analysis and sorting were performed as described previously12 (link). PBMC samples were stained by the following antibodies: CD3-Brilliant Violet 605 (clone OKT3, BioLegend), CD4-PerCP-Cy5.5 (clone OKT4, Biolegend), CCR7-PE-CF594 (clone 2-L1-A, BD Biosciences), CD27-PE (clone M-T271, BioLegend), CD45RO-APC (clone UCHL1, BioLegend). The cells were then stained by the Live-dead aqua (Invitrogen) prior to flow analysis. The stained cells were sorted on a BD FACSAria II cell sorter (BD Biosciences). Four CD4 subsets were defined as follows: naïve (CD45RO−, CCR7+, and CD27+), central memory (CD45RO+, CCR7+, and CD27+), transitional memory (CD45RO+, CCR7−, and CD27+) and effector memory (CD45RO+, CCR7−, and CD27−). The purity of each sorted CD4 subset was higher than 95%.
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