| Spleens harvested and splenocytes prepared
||Splenocytes stained and analyzed
||Populations of NK cells, NK T cells, T cells, dendritic cells
- Dissection kit
- Cell strainer (70 µM)
- Flow cytometer: BD FACS Aria II or BD LSR Fortessa X20
- Software: FlowJo
- Collagenase, Type V from Clostridium histolyticum (Sigma-Aldrich)
- Monoclonal antibodies
- CD3 (145-2C11)
- CD4 (GK1.5)
- CD8 (53-6.7)
- CD11c (N418)
- CD19 (6D5)
- CD27 (LG.3A10)
- CD49b (DX5)
- B220 (RA3-6B2)
- TCR beta (H57-597) (Biolegend)
- CD11b (M1/70) (BD Pharmingen)
- mPDCA-1 (eBio927) (eBioscience)
- mCD1d:PBS57 tetramer-PE (NIH tetramer core facility)
Procedure: Preparation of splenocytes
- 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: Splenocyte staining and analysis by flow cytometry
- Single-cell suspensions are stained with the above listed monoclonal antibodies.
- Data are acquired by flow cytometry using the following gating strategies:
- NK cell panel: Lymphocytes are gated based on FSC-A and SSC-A profile; doublets are excluded. Among singlets, NK cells are defined as CD3- CD19- CD49b+; pre-mNK are defined as B220+ CD11clow NK cells; NK functional differentiation is defined by four stages from CD27- CD11b-, CD27+ CD11b-, CD27+ CD11b+, CD27- CD11b+.
- T cell panel: Lymphocytes are gated based on FSC-A and SSC-A profile; doublets are excluded. Among singlets, NKT are defined as TCRb+ CD1d tetramer+ and T cells are defined as TCRb+ CD1d tetramer-.
- Dendritic cell (DC) panel: Dead cells and debris are excluded based on FSC-A and SSC-A profile, leaving leukocytes; doublets and autofluorescent cells are excluded. Among non-autofluorescent singlets, plasmacytoid DC (pDC) are defined as mPDCA-1+ CD11clow cells and conventional DCs (cDCs) are defined as CD11chi. Among cDCs, cDC1 are CD8a+ CD11b-, cDC2 are CD8a- CD11b+ and merocytic DC (mcDC) are defined as CD8a- CD11b-.
- Populations of NK cells, NK T cells, T cells, and dendritic cells