Cell Signaling Technology
Btk (D3H5) Rabbit mAb
8547
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The general aim of our research is to characterize the phenotype and function of pathogenic B cells in various diseases. We are building fluorescence and mass cytometry panels to study human B cells in the context of autoimmune and idiopathic pulmonary diseases. The main reason I selected BTK from Cell Signaling is because our lab uses it routinely in mice, and I wanted to test it in humans.
Flow Cytometry
Cryopreserved human PBMC
(1:200) 1uL BTK in 200uL FACS buffer with 2x10^6 cells; 30 minutes; room temperature
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(1:2000) 0.1uL anti-rabbit IgG-AF647 from Cell Signaling (Cat #4414) in 200uL FACS buffer for 20 minutes
N/A
BD LSRII 5-laser Flow Cytometer
Both the Rabbit BTK primary antibody and the anti-rabbit IgG-AF647 secondary were titrated and 1uL BTK (1:200) and 0.1uL secondary (1:2000) yielded the highest stain index. We routinely use BTK in our 14-parameter human B cell panel to identify B cells in patient samples with various diseases. Cyropreserved human PBMCs are thawed, washed twice in PBS, and ~2 million PBMC are stained with a cocktail of surface antibodies. Cells are then fixed for 5 minutes at room temperature with 1.6% paraformaldehyde, followed by a 5 minutes permeabilization on ice with 0.05% Triton-X-100. Cells are then incubated with the BTK primary antibody for 30 minutes at room temperature, washed twice, and then incubated with the secondary for 20 minutes at room temperature.
As evidenced in the attached figure, the staining looks great and the specificity of the antibody is evidenced by expression on B cells and lack of expression on most non-B cells (some innate cells can express BTK).
Staining pattern is good; specific for BTK; works in humans; works for flow cytometry
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Antibody works great!