Spleens were harvested, weighed and splenocytes isolated as previously described (24 (link)). Total splenocyte numbers were counted using a Nexcelom automatic cell counter. Lymphocyte populations were gated, (100,000 cells of the lymphocyte populations) and counted by flow cytometry. CD3+ T cell population and B220+ B cell population were counted after exclusion of the NK cell population. Antibodies were all from commercial sources and are listed below. Flow cytometry acquisition was done with a FACSCanto (BD Biosciences, San Jose, CA). Data analysis was performed using the Flowjo software (Tree Star Inc.). TCRβ-FITC, CD3-APC-Cy7, CD4-V500, CD8-AF700, B220-PECy7, CD19-PerCPCy5.5, TCRβ-PerCPCy5.5, CD4-V500 were obtained from BD Bioscience. CD11c-APC eF780 was obtained from eBioscience (eBioscience, San Diego, CA).
Protocol detail
Quantitative Splenocyte Profiling
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Corresponding Organization :
Other organizations : Office of Science, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Heart Lung and Blood Institute, Office of the Director
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Variable analysis
independent variables
- None explicitly mentioned
- Total splenocyte numbers
- Lymphocyte populations (100,000 cells)
- CD3+ T cell population
- B220+ B cell population
- Spleens were harvested, weighed and splenocytes isolated as previously described (24)
- Antibodies were all from commercial sources and are listed
- Flow cytometry acquisition was done with a FACSCanto (BD Biosciences, San Jose, CA)
- Data analysis was performed using the Flowjo software (Tree Star Inc.)
- No positive or negative controls were explicitly mentioned in the input protocol.
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