Step Description Equipment Data collected 1 Spleens harvested and splenocytes prepared Dissecting kit - 2 Splenocytes stained and analyzed Flow cytometer total number of splenocytes, total number of B cells in spleen, percentage of splenocytes that are B cells
General Information for All Procedures: Data for Lesage1 and Lesage2 were collected at the same time. Lesage1 contains populations of NK cells, NK T cells, T cells, and dendritic cells while Lesage2 contains data about B cells.
Procedure 1: Preparation of splenocytes
Equipment, software, and supplies
- Dissection kit
- Cell strainer (70 µM)
- Centrifuge
Reagents and solutions
- Collagenase, Type V from Clostridium histolyticum (Sigma-Aldrich)
Steps
- Spleens are harvested and treated with collagenase (1 mg/mL in PBS) for 15 min at 37°C.
- Cells are passed through a cell strainer (70 µM) to yield single-cell suspensions prior to staining with antibodies.
Procedure 2: Splenocyte staining and analysis by flow cytometry
Equipment, software, and supplies
- Flow cytometer: BD FACS Aria II or BD LSR Fortessa X20 (Becton-Dickinson)
- Software: FlowJo
Reagents and solutions
- Monoclonal antibody CD3 (145-2C11)
- Monoclonal antibody B220 (RA3-6B2)
- Monoclonal antibody CD19 (6D5)
- Monoclonal antibody CD49b (DX5)
Steps
- Single-cell suspensions are stained with the above listed monoclonal antibodies.
- Data are acquired by flow cytometry using the following gating strategies: B cells: CD3-CD49b-B220+CD19+
Primary References
Aubin AM, Vdovenko D, Collin R, Balmer L, Coderre L, Morahan G, Lombard-Vadnais F, Lesage S. Variations in the germinal center response revealed by genetically diverse mouse strains. Immunol Cell Biol. 2024 Nov;102(10):935-948. doi: 10.1111/imcb.12823. Epub 2024 Sep 24.
PubMed 39318030 Collin R, Balmer L, Morahan G, Lesage S. Common Heritable Immunological Variations Revealed in Genetically Diverse Inbred Mouse Strains of the Collaborative Cross. J Immunol. 2019 Feb 1;202(3):777-786. doi: 10.4049/jimmunol.1801247. Epub 2018 Dec 26.
PubMed 30587532