1 Introduction

The largest organ of the lymphatic system, the spleen, is responsible for initiating immune reactions to blood-borne antigens and for filtering blood of foreign material and old or damaged blood cells. These processes take place in the two main compartments of the spleen: the white pulp with its marginal zone and the red pulp. In alignment with their different functions, the two compartments vary in morphology and cellular composition.

The red pulp is a blood filter. It controls the amount of red blood cells in the body and stores erythrocytes and platelets. Healthy erythrocytes pass through narrow passages within the spleen, while old and damaged erythrocytes are removed and digested to recycle iron and protein components of hemoglobin. In rodents, the red pulp is a site of hematopoiesis.

The white pulp consists of lymphocytes, macrophages, dendritic cells, and plasma cells. The immune cells destroy filtered pathogens and produce respective antibodies. During infections, the spleen might enlarge due to an increased number of white blood cells.

2 Cell types, frequencies, and marker expression

At a glance: Cell types in spleen tissue

Cell type or subsetFrequency (% of total cells)MarkersFunction
T cells21–25CD90, CD3, CD4, CD8Adaptive immune response
B cells44–58CD19, CD45R/B220
  • Antibody and cytokine secretion
  • Antigen presentation
Monocytes3.5–5CD11b, CD115, CCR2, Ly6C
  • Migrate to site of infection
  • Differentiate into macrophages or dendritic cells
Granulocytes1–2CD11b, Ly6C, SiglecF, CD49b, CD193
  • Among first responders at site of inflammation
  • Pathogen destruction
  • Recruitment of immune cells
Dendritic cells1–3CD11c, MHCII, XCR1, Tissue-based sentinels that activate the immune response
Natural killer cells1–2NKp46, NK1.1Recognize and destroy infected cells without antigen presentation on MHC
Marcophages1–2F4/80Phagocytosis of cellular debris, microbes, cancer cells, etc.
Given its role in the lymphatic system, the spleen hosts a broad spectrum of blood cells, both erythrocytes and leukocytes, as well as dead cells, cell debris, and cell fragments (platelets). In terms of understanding cell subpopulations involved in immune reactivity and the mechanisms of their function, spleen is an excellent source of several immune cell subtypes.

3 Sample preparation

At a glance: Kits, reagents, and hardware for the preparation of spleen samples

Kits and reagents
Dissociation of spleen tissueEnsures cell surface epitope preservation. Used with gentleMACS Dissociator and C Tubes, is gentlest method to obtain a single-cell suspensions without compromising cell yield or function.Spleen Dissociation Kit, mouse
Hardware and consumables
Tissue dissociationPre-set programs to dissociate different tissues with Miltenyi Biotec 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.MACS SmartStrainer, 70 µm

Lymphocytes are easily released from spleen for subsequent isolation by shearing the tissue using a MACS® SmartStrainer, 70 µm. To isolate tissue-resident cells however, such as dendritic cells and macrophages, the tissue must be broken down enzymatically and mechanically to release the infiltrating 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 cytometry analysis. The use of validated enzymes ensures reproducibility and quality of the tissue dissociation for downstream experiments.

Combining mechanical dissociation by using a gentleMACS™ Dissociator and gentleMACS C Tubes together with the Spleen Dissociation Kit, mouse quickly and easily generates single-cell suspensions from mouse spleen 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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