{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T03:45:30Z","timestamp":1773114330088,"version":"3.50.1"},"reference-count":22,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,9,1]],"date-time":"2025-09-01T00:00:00Z","timestamp":1756684800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1617203","type":"journal-article","created":{"date-parts":[[2025,9,1]],"date-time":"2025-09-01T05:30:15Z","timestamp":1756704615000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Comparative evaluation of isolation techniques and characterization of red pulp macrophages from pig splenocytes"],"prefix":"10.3389","volume":"16","author":[{"given":"Phu Chi","family":"Vu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nhat Minh","family":"Dang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonghyeok","family":"Jung","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min Hyuk","family":"Kim","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min Guk","family":"Lee","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joohyun","family":"Shim","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thi Xoan","family":"Hoang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jae Young","family":"Kim","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,9,1]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1606751113","article-title":"Biomechanics of red blood cells in human spleen and consequences for physiology and disease","volume":"113","author":"Pivkin","year":"2016","journal-title":"Proc Natl Acad Sci"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2017.00033","article-title":"The heme connection: linking erythrocytes and macrophage biology","volume":"8","author":"Alam","year":"2017","journal-title":"Front Immunol"},{"key":"B3","doi-asserted-by":"publisher","first-page":"129","DOI":"10.1038\/s42003-024-05802-9","article-title":"A secretory protein neudesin regulates splenic red pulp macrophages in erythrophagocytosis and iron recycling","volume":"7","author":"Nakayama","year":"2024","journal-title":"Commun Biol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1038\/s41375-018-0207-9","article-title":"Dysregulated iron metabolism in polycythemia vera: etiology and consequences","volume":"32","author":"Ginzburg","year":"2018","journal-title":"Leukemia"},{"key":"B5","doi-asserted-by":"publisher","first-page":"e90189","DOI":"10.7554\/eLife.90189","article-title":"Normal and dysregulated crosstalk between iron metabolism and erythropoiesis","volume":"12","author":"Ginzburg","year":"2023","journal-title":"Elife"},{"key":"B6","doi-asserted-by":"publisher","first-page":"492","DOI":"10.1016\/j.immuni.2016.02.016","article-title":"Macrophages and iron metabolism","volume":"44","author":"Soares","year":"2016","journal-title":"Immunity"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1038\/nri1669","article-title":"Structure and function of the spleen","volume":"5","author":"Mebius","year":"2005","journal-title":"Nat Rev Immunol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1159\/000336423","article-title":"Macrophages and systemic iron homeostasis","volume":"4","author":"Ganz","year":"2012","journal-title":"J Innate Immun"},{"key":"B9","first-page":"535","article-title":"Tissue distribution of IgG Fc receptors","volume":"47","author":"Schrieber","year":"1982","journal-title":"Clin Exp Immunol"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2011.04.076","article-title":"Delivery of antigen to sialoadhesin or CD163 improves the specific immune response in pigs","volume":"29","author":"Poderoso","year":"2011","journal-title":"Vaccine"},{"key":"B11","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1016\/j.vetmic.2016.12.004","article-title":"Splenic CD163+ macrophages as targets of porcine reproductive and respiratory virus: role of Siglecs","volume":"198","author":"Yuste","year":"2017","journal-title":"Vet Microbiol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1182\/bloodadvances.2017015008","article-title":"Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-\u03b3 receptors","volume":"2","author":"Nagelkerke","year":"2018","journal-title":"Blood Adv"},{"key":"B13","doi-asserted-by":"publisher","first-page":"597","DOI":"10.1189\/jlb.0310184","article-title":"Technical advance: autofluorescence as a tool for myeloid cell analysis","volume":"88","author":"Mitchell","year":"2010","journal-title":"J Leuk Biol"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1016\/j.dci.2014.03.022","article-title":"The porcine innate immune system: an update","volume":"45","author":"Mair","year":"2014","journal-title":"Dev Comp Immunol"},{"key":"B15","doi-asserted-by":"publisher","first-page":"e2025428118","DOI":"10.1073\/pnas.2025428118","article-title":"Human-relevant near-organ neuromodulation of the immune system via the splenic nerve","volume":"118","author":"Doneg\u00e0","year":"2021","journal-title":"Proc Natl Acad Sci"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1373\/clinchem.2008.112797","article-title":"The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments","volume":"55","author":"Bustin","year":"2009"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1038\/nature07472","article-title":"Role for Spi-C in the development of red pulp macrophages and splenic iron homeostasis","volume":"457","author":"Kohyama","year":"2009","journal-title":"Nature"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1038\/srep12940","article-title":"Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates","volume":"5","author":"Franken","year":"2015","journal-title":"Sci Rep"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1038\/s41418-022-00932-1","article-title":"Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia","volume":"29","author":"Vallelian","year":"2022","journal-title":"Cell Death Different"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1086\/340213","article-title":"Central role of the spleen in malaria parasite clearance","volume":"185","author":"Chotivanich","year":"2002","journal-title":"J Infect Dis"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2015.00480","article-title":"Splenic macrophage subsets and their function during blood-borne infections","volume":"6","author":"Borges da Silva","year":"2015","journal-title":"Front Immunol"},{"key":"B22","doi-asserted-by":"publisher","first-page":"946","DOI":"10.1182\/blood.2020005351","article-title":"Hemolysis in the spleen drives erythrocyte turnover","volume":"134","author":"Klei","year":"2019","journal-title":"Blood"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1617203\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,1]],"date-time":"2025-09-01T05:30:16Z","timestamp":1756704616000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1617203\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,1]]},"references-count":22,"alternative-id":["10.3389\/fimmu.2025.1617203"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1617203","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,1]]},"article-number":"1617203"}}