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Updated: May 5, 2026
Isolation of Murine Lymph Node Stromal Cells
Published on: August 19, 2014
Dissociation protocols influence the phenotypes of lymphocyte and myeloid cell populations isolated from the neonatal
Jarina P DaMata1,2, Amanda E Zelkoski1, Paula B Nhan1,2
1Laboratory of Infectious Diseases and Host Defense, Department of Pediatrics, Uniformed Services University of Health Sciences (USUHS), Bethesda, MD, United States.
Insights
Investigating immune cell isolation from lymph nodes (LN) in neonatal and adult mice revealed that while cell yield and viability were similar across three dissociation methods, enzymatic digestion altered key immune cell markers, impacting experimental results.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- Immune cell frequencies and phenotypes differ between neonates and adults, necessitating age-specific immune response studies.
- Lymph nodes (LN) are crucial for quantifying these age-related immune differences.
- Tissue processing methods can significantly impact immune cell analysis, potentially influencing experimental outcomes.
Purpose of the Study:
- To compare the impact of three different dissociation protocols on immune cell isolation from mouse lymph nodes.
- To assess how these protocols affect cell yield, viability, and phenotypic/maturation markers in neonatal and adult mice post-respiratory syncytial virus (RSV) infection.
Main Methods:
- Compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I, and mechanical dissociation with density gradient separation.
- Analyzed immune cells from lung-draining lymph nodes of neonatal and adult mice two days after intranasal RSV infection.
- Evaluated cell yield, viability, and expression of phenotypic and activation markers using flow cytometry.
Main Results:
- All three dissociation protocols yielded comparable cell numbers and viability in both neonatal and adult lymph nodes.
- Enzymatic digestion did not increase the yield of conventional dendritic cells and macrophages compared to mechanical methods.
- Enzymatic digestion significantly altered the mean fluorescence intensity of key lineage and activation markers.
Conclusions:
- Dissociation protocols have similar effects on the quantity and viability of immune cells isolated from neonatal and adult lymph nodes.
- Enzymatic digestion can impact the measured expression levels of immune cell markers, potentially affecting downstream experimental interpretations.
- Careful selection of tissue dissociation methods is crucial for accurate characterization of immune cell populations, especially when comparing age groups or experimental conditions.
Abstract:
Frequencies and phenotypes of immune cells differ between neonates and adults in association with age-specific immune responses. Lymph nodes (LN) are critical tissue sites to quantify and define these differences. Advances in flow cytometry have enabled more multifaceted measurements of complex immune responses. Tissue processing can affect the immune cells under investigation that influence key findings. To understand the impact on immune cells in the LN after processing for single-cell suspension, we compared three dissociation protocols: enzymatic digestion, mechanical dissociation with DNase I treatment, and mechanical dissociation with density gradient separation. We analyzed cell yields, viability, phenotypic and maturation markers of immune cells from the lung-draining LN of neonatal and adult mice two days after intranasal respiratory syncytial virus (RSV) infection. While viability was consistent across age groups, the protocols influenced the yield of subsets defined by important phenotypic and activation markers. Moreover, enzymatic digestion did not show higher overall yields of conventional dendritic cells and macrophages from the LN. Together, our findings show that the three dissociation protocols have similar impacts on the number and viability of cells isolated from the neonatal and adult LN. However, enzymatic digestion impacts the mean fluorescence intensity of key lineage and activation markers that may influence experimental findings.
