Lesage1 project protocol

Immunological variation in 68 Collaborative Cross strains of mice and 8 founder strains   (2018)

Lesage S, Collin R, Balmer L, Morahan G

See also: Lesage1 animal documentation


Project protocol - Contents

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+ 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-.

Data collected by investigator

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