The human nasopharyngeal samples were processed immediately post-surgery. The tissue was minced, treated with 0.25 mg/ml of collagenase (Sigma-Aldrich, St. Louis, MO) at 37°C for 60 minutes and cell isolation was performed. Cells were surface stained with CD45, CD20, CD3, CD335, CD66b, BDCA-1, BDCA-2, BDCA-3, CD14, and Live/Dead Fixable Blue Dead Cell Stain (Life technologies, Grand Island, NY). Titrated Abs were added to the cells in 50 μl PBS 1% FCS for 30 min at 4°C. Washed cells were fixed in 2% formaldehyde and stored at 4°C until analysis, which was performed using an LSR II flow cytometer (BD Biosciences, San Jose, CA). The whole sample was acquired and analysis was preformed using Flow Jo 9.1 software (Tree Star, Ashland, OR). To identify human DC subsets, dead cells were excluded and CD3+, CD20+, CD335+, CD66b+, and CD14+ cells i.e., T, B, NK, neutrophils and monocytes were sequentially gated out; three DC populations were identified within the CD45+ mononuclear cells by gating respectively on either BDCA-1+ cells, BDCA-2+ cells or BDCA-3hi cells.
Bdca 3
Manufactured by Thermo Fisher Scientific
The BDCA-3 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for the detection and quantification of specific biological targets, such as proteins or nucleic acids, in a sample. The BDCA-3 utilizes advanced detection techniques to provide reliable and accurate results. Further details on the specific intended use or functionalities of this product are not available.
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2 protocols using bdca 3
1
Characterization of Human Nasal Dendritic Cells
2
Characterization of Human Nasal Dendritic Cells
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The human nasopharyngeal samples were processed immediately post-surgery. The tissue was minced, treated with 0.25 mg/ml of collagenase (Sigma-Aldrich, St. Louis, MO) at 37°C for 60 minutes and cell isolation was performed. Cells were surface stained with CD45, CD20, CD3, CD335, CD66b, BDCA-1, BDCA-2, BDCA-3, CD14, and Live/Dead Fixable Blue Dead Cell Stain (Life technologies, Grand Island, NY). Titrated Abs were added to the cells in 50 μl PBS 1% FCS for 30 min at 4°C. Washed cells were fixed in 2% formaldehyde and stored at 4°C until analysis, which was performed using an LSR II flow cytometer (BD Biosciences, San Jose, CA). The whole sample was acquired and analysis was preformed using Flow Jo 9.1 software (Tree Star, Ashland, OR). To identify human DC subsets, dead cells were excluded and CD3+, CD20+, CD335+, CD66b+, and CD14+ cells i.e., T, B, NK, neutrophils and monocytes were sequentially gated out; three DC populations were identified within the CD45+ mononuclear cells by gating respectively on either BDCA-1+ cells, BDCA-2+ cells or BDCA-3hi cells.
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