dLNs (2 inguinal, 2 axillary, 2 brachial, 2 popliteal, and 4 cervical LNs per mouse) were isolated and digested as described (45 (link)) in RPMI 1640 with 2% FBS, 0.8 mg/ml Dispase (Roche Diagnostics), 0.2 mg/ml Collagenase P (Roche Diagnostics), and 0.1 mg/ml DNase I (Sigma-Aldrich). Single cell suspensions were stained in the presence of Fc-block with fluorochrome-conjugated antibodies (all purchased from BioLegend unless otherwise indicated) against the following targets: CD4 (RM4-4), CD45 (30-F11), CD3 (1452C11), TCRb (H57-597), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), gp38 (8.1.1), CD31 (MEK13.3), S1PR1 (713412, R&D Systems). Staining with S1PR1 antibody was performed in the presence of fatty acid–free BSA from Sigma-Aldrich; blood cells were used as S1PR negative controls (17 (link), 46 (link)). Stained cells were analyzed with the FACS Canto II (BD Biosciences) and Flow-Jo software. For ki67 (clone 16A8, BioLegend) intracellular staining, cells were fixed and permeabilized with the Foxp3 Fix/Perm Buffer Set (BioLegend). LN stromal cells were sorted as described (45 (link)) using a BD FACSAria Fusion cell sorter; CD45+ cells were depleted prior to sorting with anti-biotin magnetic beads (Miltenyi Biotec).
Protocol detail
Isolation and Analysis of Lymph Node Cells
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Dispase
Collagenase P
DNase I
fluorochrome-conjugated antibodies
), S1PR1
FACS Canto II
Foxp3 Fix/Perm Buffer Set
BD FACSAria Fusion cell sorter
anti-biotin magnetic beads
Antibodies
Antibody
Axillary
Biotin
Blood cells
Buffer
Cd44
Cd62l
Cells
Cervical
Clone
Collagenase
Diagnostics
Dispase
Dnase
Fluorochrome
Free fatty acid
Inguinal
Intracellular
Mouse
Perm
Stromal cells
Tcrb
Corresponding Organization : Harvard University
Other organizations : Dana-Farber Cancer Institute, Boston University, Ragon Institute of MGH, MIT and Harvard
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Variable analysis
independent variables
- Dispase concentration (0.8 mg/ml)
- Collagenase P concentration (0.2 mg/ml)
- DNase I concentration (0.1 mg/ml)
- Characteristics of isolated lymph node stromal cells (e.g., cell surface marker expression, proliferation)
- RPMI 1640 media with 2% FBS
- Lymph node types (2 inguinal, 2 axillary, 2 brachial, 2 popliteal, and 4 cervical per mouse)
- Antibodies used for staining (CD4, CD45, CD3, TCRb, CD44, CD62L, CD69, gp38, CD31, S1PR1, Ki67)
- Blood cells used as S1PR1 negative controls
- None mentioned
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