Abstract
Our protocol describes a simple procedure for isolating stromal cells from lymph nodes (LN). LN are disrupted then enzymatically digested with collagenase and dispase to produce a single cell suspension that can be stained with fluorescently labelled antibodies and analysed by flow cytometry. This protocol will enable identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC) as PNAd+ BEC that form LN high endothelial venules (HEV). This method can be applied to examine LN stromal cell responses during inflammatory events induced by infections or immunologic adjuvants and to subset most leukocytes found in LN.
Keywords: Lymphoid stromal cells, Lymph nodes, Endothelial cells, Fibroblastic reticular cells, Immune response, Virus infection
Background
Lymph nodes (LN) are constructed of complex networks of mesenchymal and endothelial stromal cells. These include the fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs) and blood endothelial cells (BECs). These stromal cells organize the complex microarchitecture of LN, enabling the support of immune cell migration, homeostasis, tolerance and cellular interactions required for the initiation of immune responses to pathogens and tumors. We have shown that the LN stromal cells can proliferate and expand in response to inflammatory signals and the recruitment of immune cells into LN that accompanies infections (Gregory et al., 2017). These stromal cells can also significantly modulate their transcriptional program to respond to infection, thereby supporting ongoing immune responses. This protocol enables reliable isolation of stromal cell subsets from LN both in the steady state and during disease. This enables phenotypic, functional, genetic or epigenetic investigation of LN stromal cells to reveal how they contribute to tissue homeostasis and immune responses.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
It is recommended to include 4-5 mice per group and perform 2-3 independent experiments. Flow cytometry files are analysed using FlowJo to gate and quantitate individual stromal cells subsets (Figure 1). To analyse LN stromal cell subsets during infection, draining and non-draining LN are compared, and populations of stromal cells quantitated and numbers plotted (Figure 2). This procedure will also release all leukocytes from the LN, enabling additional antibody staining and analysis of other cell types if required (see Note 1). To assess statistical significance between groups, unpaired t-tests or Mann-Whitney tests should be performed, as appropriate, using Prism (GraphPad). Figure 1. Gating strategy used to identify LN stromal cell subsets. Dead cells and doublet cells are first excluded. Stromal cells lack CD45.2 and Ter119, markers of hematopoietic cells and erythroid cells, respectively. The use of gp38 and CD31 allows the identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC) and blood endothelial cells (BEC). The marker PNAD is used to identify the BEC that form the high endothelial venules (HEV) in LN. Bar graphs show the quantification of the stromal cell subsets in the brachial LN (bLN), inguinal LN (iLN) and the popliteal LN (pLN) at steady state. Figure 2. Analysis of stromal cell subsets in draining brachial LN and non-draining inguinal LN by flow cytometry after Herpes simplex virus (HSV) infection. Mice were infected by scarification on the left flank skin with HSV and LN harvested 7 days post-infection for stromal cell analysis. During an immune challenge, stromal cells expand to accommodate the increase of total cellularity in the LN.
Notes
Dispase treatment can cleave certain cell surface markers. We found that typical markers of stromal cells are not affected by our enzymatic digestion method. We routinely analyse leukocyte subsets in the LN and found that CD8α and CD19 staining is diminished when this protocol is applied. The use of CD8β and B220 as alternative markers is recommended.
Recipes
Acknowledgments
This protocol was adapted from our publication (Gregory et al., 2017). This work was supported by the Australian Research Council.
Competing interests
The authors declare that they have no competing interests.
References
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