Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Preparation of splenocytes
• Procedure: Splenocyte staining and analysis by flow cytometry
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Spleens harvested and splenocytes prepared	Dissecting kit	-
2	Splenocytes stained and analyzed	Flow cytometer	Populations of NK cells, NK T cells, T cells, dendritic cells

Equipment and supplies

• Dissection kit
• Cell strainer (70 µM)
• Centrifuge
• Flow cytometer: BD FACS Aria II or BD LSR Fortessa X20
• Software: FlowJo

Reagents and solutions

• 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

1. Spleens are harvested and treated with collagenase (1 mg/mL in PBS) for 15 min at 37°C.
2. 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

1. Single-cell suspensions are stained with the above listed monoclonal antibodies.
2. 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+ CD11c^low 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+ CD11c^low cells and conventional DCs (cDCs) are defined as CD11c^hi. Among cDCs, cDC1 are CD8a+ CD11b-, cDC2 are CD8a- CD11b+ and merocytic DC (mcDC) are defined as CD8a- CD11b-.

Data collected by investigator

• Populations of NK cells, NK T cells, T cells, and dendritic cells