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Comparative evaluation of isolation techniques and characterization of red pulp macrophages from pig splenocytes
Phu Chi Vu1, Nhat Minh Dang1, Jonghyeok Jung1
1Department of Life Science, Gachon University, Seongnam, Kyeonggi-Do, Republic of Korea.
Introduction:
Red pulp macrophages (RPMs) play a central role in iron recycling and immune regulation within the spleen, yet optimized methods for the isolation and characterization of pig RPM remain limited.
Methods:
We compared two approaches for isolating RPMs from pig splenocytes: CD163 antibody- based sorting and magnetic-activated cell sorting (MACS), which leverages the natural iron content and autofluorescence of RPMs. Isolated cells were evaluated by flow cytometry for marker expression, and functional assays were performed to assess phagocytic activity and gene expression related to iron metabolism.
Results:
Flow cytometry identified an autofluorescent population, a hallmark of RMPs, within the pig splenocytes. CD163-based method enriched RPMs to 71.8% autofluorescent cells, while the MACS- based approach achieved a higher yield of 81% autofluorescent cells without using antibodies, demonstrating greater cost-effectiveness and efficiency. Marker analysis revealed high expression of CD16 and CD163, moderate expression of CD11b, and low or undetectable levels of CD14, CD32, and CD169. Functionally, isolated RPMs demonstrated robust phagocytosis of senescent red blood cells and upregulation of genes involved in heme and iron metabolism.
Discussion:
These findings establish an optimized, antibody-free protocol for efficient isolation of pig RPMs. The approach provides a reliable platform for studying splenic macrophage biology, iron homeostasis, and immunological research and splenic function studies.
Insights
We developed an efficient, antibody-free method to isolate pig red pulp macrophages (RPMs) using magnetic-activated cell sorting (MACS). This technique enhances studies on spleen immunity and iron recycling in pigs.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Red pulp macrophages (RPMs) are crucial for iron recycling and immune regulation in the spleen.
- Optimized methods for isolating and characterizing pig RPMs are limited, hindering research.
Purpose of the Study:
- To compare CD163 antibody-based sorting with magnetic-activated cell sorting (MACS) for isolating pig RPMs.
- To establish an efficient, antibody-free protocol for pig RPM isolation and characterization.
Main Methods:
- Two methods were compared: CD163 antibody-based sorting and MACS, leveraging RPM autofluorescence and iron content.
- Isolated cells were analyzed via flow cytometry for marker expression (CD16, CD163, CD11b, CD14, CD32, CD169).
- Functional assays assessed phagocytosis of senescent red blood cells and gene expression related to iron metabolism.
Main Results:
- MACS yielded a higher percentage (81%) of autofluorescent RPMs compared to the CD163 method (71.8%).
- MACS is more cost-effective and efficient, avoiding antibody use.
- Isolated RPMs exhibited high CD16 and CD163 expression, robust phagocytic activity, and upregulated iron metabolism genes.
Conclusions:
- An optimized, antibody-free MACS protocol for pig RPM isolation was established.
- This method provides a reliable platform for studying splenic macrophage biology, iron homeostasis, and immune function in pigs.
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