For wild type (WT), DβYTL and Dβ2ko mice, single cell suspensions were prepared from the thymus of two mice each. Red blood cells were removed with RBC lysing solution (1 mM KHCO3, 0.15 M NH4Cl, and 0.1 mM Na2EDTA). Cells were washed twice and resuspended in a master-mix of staining buffer containing optimal concentrations of monoclonal antibody reagents. Samples were sorted with a FACS Aria (Becton Dickinson). Double negative thymic cells were stained with PE-Cy7-CD25 (BD Cat #552880), APC-CD44 (BD Cat #559250), biotinylated-CD28 (BD Cat #553296) (developed secondarily with streptavidin), and a lineage stain [PE-CD3 (BD Cat #555275), monoclonal PE-CD4 (BD Cat #553049), PE-CD8α (BD Cat #553033), PE-B220 (BD Cat #561878), PE-CD11b (BD Cat #553311), PE-NK1.1 (BD Cat#553165)] to remove mature T, B, and NK cells. The cells were stained with propidium iodide (PI) to identify/sort live cells. DN2 cells were defined as CD44+ and CD25+ (Supplementary Figure S2 ).
Biotinylated cd28
Manufactured by BD
Biotinylated-CD28 is a laboratory reagent that consists of the CD28 protein chemically conjugated to biotin. CD28 is a cell surface receptor that provides a costimulatory signal to T cells, enhancing their activation and proliferation. The biotinylation of CD28 allows for its detection and purification through binding to streptavidin-based systems.
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Lab products found in correlation
Facsaria, by BD (2 mentions)
Cd4 pe, by BD (2 mentions)
Cd11b pe, by BD (2 mentions)
Cd25 pe cy7, by BD (2 mentions)
Cd44 apc, by BD (2 mentions)
Cd3 pe, by BD (2 mentions)
Cd8α pe, by BD (2 mentions)
B220 pe, by BD (2 mentions)
Facs lsr 2, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Apc cd8α, by BD (1 mentions)
2 protocols using biotinylated cd28
1
Isolation of Murine Thymic Double-Negative T Cells
2
Immunophenotyping Murine Thymus and Spleen
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Single cell suspensions were prepared from both the thymus and spleen of two mice. Red blood cells were removed with RBC lysing solution (1 mM KHCO3, 0.15 M NH4Cl, and 0.1 mM Na2EDTA). Cells were washed twice and resuspended in a master-mix of staining buffer containing optimal concentrations of monoclonal antibody reagents. Samples were analyzed on a FACS LSR II (Becton Dickinson) or sorted with a FACS Aria (Becton Dickinson). Double negative thymic cells were stained with PE-Cy7- CD25 (BD Cat#552880), APC- CD44 (BD Cat#559250), biotinylated- CD28 (BD Cat#553296) (developed secondarily with streptavidin), and a lineage stain [PE-CD3 (BD Cat#555275), monoclonal PE-CD4 (BD Cat#553049), PE-CD8α (BD Cat#553033), PE-B220 (BD Cat#561878), PE-CD11b (BD Cat#553311), PE-NK1.1 (BD Cat#553165)] to remove mature T, B, and NK cells. Double and single positive thymic cells were stained with PE-CD3 (BD Cat#555275), FITC-CD4 (BD Cat#557307), and APC-CD8α (BD Cat#557682). Splenic cells were stained with PE-CD3 (BD Cat#555275), FITC-CD4 (BD Cat#557307), and APC-CD8α (BD Cat#557682). All subsets were also stained with propidium iodide (PI) to exclude dead cells.
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