MACS Handbook

Lymph node

1 Introduction

Lymph nodes are ovoid-shape organs found throughout the lymphatic system, a network of vessels that collects interstitial fluid surrounding the cells of the body and transports it back to the circulatory system. The collected interstitial fluid, known as lymph, passes through lymph nodes where it is screened for antigens that can activate immune responses. Thus, lymph nodes can become hypertrophic during an infection, drug reaction, autoimmune disease, or cancer.

A capsule of dense connective tissue surrounds an inner network of sinuses through which lymph flows. Lymphoid tissue is located among these sinuses and presents three distinct regions: cortex, paracortex, and medulla (PMID: 15039048; Monographs: Janeway’s Immunology, 7th Edition, Hoffbrand’s Essential Haematology, 7th Edition).

2 Cell types, frequencies, and marker expression

At a glance: Cell types in lymph node tissue (PMID: 25178108, 28207940, 22488251)

Cell type or subset

Frequency (%)

of total cells
MarkersFunction
Stromal cells 0.3–0.5 CD31, PDPN, MadCAM
  • Supporting role
  • Expression of surface molecules
  • Chemokine production
Dendritic cells0.5–1CD11c, MHCII, XCR1
  • Cytokine and chemokine production
  • Antigen presentation
B cells9–15 CD19, CD45R/B220
  • Antibody and cytokine secretion
  • Antigen presentation
  • Memory responses
T cells65–78 CD90, CD3, CD4, CD8
  • Cytokine secretion
  • Induction of isotype switching
  • Target cell lysis
  • Memory responses
Macrophages1–2CD169, CD11b, F4/80
  • Tissue-based phagocytes
  • Antigen presentation

Two major cellular components can be distinguished in the cortex, paracortex, and medulla of lymph nodes. The stroma is formed from fixed cell populations of stroma cells, whereas the parenchyma consists of migrating cell populations, such as lymphocytes and dendritic cells.

The migrating cell populations enter lymph nodes through high endothelial veins attracted by chemokines, and distribute differently within the inner regions. B cells localize to the cortex, where they form primary and secondary follicles. T cells accumulate in the paracortex where they encounter dendritic cells that drive their activation. After activation, effector T cells promote terminal B cell differentiation into plasma cells that then migrate towards the medulla. Eventually, plasma cells and effector T cells leave the lymph node through the intermediate and medullary sinuses and the efferent lymphatic vessel, and return to the bloodstream. Naive lymphocytes that do not recognize their antigen also leave through the efferent lymphatic vessel and return to blood where they continue to recirculate through lymphoid tissues until they recognize an antigen or die.

3 Sample preparation

At a glance: Kits, reagents, and hardware for the preparation of mouse lymph nodes 

UseCommentsProduct
Kits and reagents
Dissociation of lymph node tissueThis kit can be used for mouse lymph nodes as well. Ensures preservation of cell surface epitopes. Used with gentleMACS Dissociator and C Tubes, it is the gentlest method to obtain single-cell suspensions without compromising cell yield or function.Spleen Dissociation Kit, mouse
Hardware and consumables
Tissue dissociationProvides pre-set programs to dissociate different tissues with Miltenyi Biotec's enzyme kits for high cell yield and viability.gentleMACS Octo Dissociator with Heaters
Tissue dissociationPrecision tool coupled with gentleMACS Dissociators that generates optimal shearing to efficiently disrupt tissue while keeping cells intact.gentleMACS C Tube
FiltrationSmart design prevents clogging, enables one-step filtering with decreasing filter sizes, and accommodates 50 mL or 15 mL tubes.SmartStrainer, 100 µm

Lymphocytes are easily released from lymph nodes for subsequent isolation by shearing the tissue using a MACS® SmartStrainer, 100 µm. To isolate stroma cells and tissue-resident cells however, such as dendritic cells and macrophages, the tissue must be broken down enzymatically to release these cells.

Crude digestion enzymes vary in their specific activities. Depending on the enzymes used, cleavage of cell surface epitopes can occur, which impacts the performance of downstream experiments, including cell isolation, flow sorting, and flow analysis. The use of validated enzymes ensures reproducibility and quality of the tissue dissociation for downstream experiments.

The Spleen Dissociation Kit, mouse can be used for the dissociation of lymph nodes from mice. The combination of this enzyme kit with mechanical dissociation using the gentleMACS™ Dissociator and gentleMACS C Tubes quickly and easily generates single-cell suspensions from mouse lymph nodes with high numbers of leukocytes, including dendritic cells. Moreover, the lot-to-lot consistency of the enzymes and automation of the mechanical dissociation step ensure reproducible results every time and high-quality downstream analyses.
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Sample preparation

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