Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 Software
In vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, p...
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Bio-protocol LLC
2025-06-01
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| author | Marlene Bravo-Parra Rahul Nelli Lu Yen Gino Castillo Juan Mora-Díaz Ana Castillo-Espinoza Luis Giménez-Lirola |
| author_facet | Marlene Bravo-Parra Rahul Nelli Lu Yen Gino Castillo Juan Mora-Díaz Ana Castillo-Espinoza Luis Giménez-Lirola |
| author_sort | Marlene Bravo-Parra |
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| description | In vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, peripheral blood mononuclear cells (PBMCs) offer more accessible and practical sampling. However, a streamlined protocol for porcine PBMCs proliferation with robust batch analysis has been lacking. We therefore developed a detailed workflow for inducing proliferation in cryopreserved porcine PBMCs using 5 μg/mL concanavalin A (ConA). The protocol covers cell isolation, cryopreservation, ConA stimulation, CD4+ T-cell staining, flow cytometry acquisition and gating on an Attune NxT instrument, and batch analysis with FCS ExpressTM 7.18. This approach yielded 78.9% viable cells, of which 33.8% were CD4+ lymphocytes. Moreover, 93.9% (n = 216) of cells proliferated, yielding up to nine cell generations. Batch analysis in FCS ExpressTM enhanced the accuracy and interpretation of proliferation metrics. This validated protocol provides a reliable framework for generating consistent proliferation data in porcine immunology studies. |
| format | Article |
| id | doaj-art-7acb33c39e57436ca59cab962a3e9864 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Bio-protocol LLC |
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| series | Bio-Protocol |
| spelling | doaj-art-7acb33c39e57436ca59cab962a3e98642025-08-20T03:32:23ZengBio-protocol LLCBio-Protocol2331-83252025-06-01151110.21769/BioProtoc.5332Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 SoftwareMarlene Bravo-Parra0Rahul Nelli1Lu Yen2Gino Castillo3Juan Mora-Díaz4Ana Castillo-Espinoza5Luis Giménez-Lirola6Department of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1850 Christensen Drive, Ames, IA, USADepartment of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1850 Christensen Drive, Ames, IA, USADepartment of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1850 Christensen Drive, Ames, IA, USADepartment of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1850 Christensen Drive, Ames, IA, USADepartment of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1850 Christensen Drive, Ames, IA, USADepartment of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1850 Christensen Drive, Ames, IA, USADepartment of Veterinary Diagnostic & Production Animal Medicine, College of Veterinary Medicine, Iowa State University, 1850 Christensen Drive, Ames, IA, USAIn vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, peripheral blood mononuclear cells (PBMCs) offer more accessible and practical sampling. However, a streamlined protocol for porcine PBMCs proliferation with robust batch analysis has been lacking. We therefore developed a detailed workflow for inducing proliferation in cryopreserved porcine PBMCs using 5 μg/mL concanavalin A (ConA). The protocol covers cell isolation, cryopreservation, ConA stimulation, CD4+ T-cell staining, flow cytometry acquisition and gating on an Attune NxT instrument, and batch analysis with FCS ExpressTM 7.18. This approach yielded 78.9% viable cells, of which 33.8% were CD4+ lymphocytes. Moreover, 93.9% (n = 216) of cells proliferated, yielding up to nine cell generations. Batch analysis in FCS ExpressTM enhanced the accuracy and interpretation of proliferation metrics. This validated protocol provides a reliable framework for generating consistent proliferation data in porcine immunology studies.https://bio-protocol.org/en/bpdetail?id=5332&type=0 |
| spellingShingle | Marlene Bravo-Parra Rahul Nelli Lu Yen Gino Castillo Juan Mora-Díaz Ana Castillo-Espinoza Luis Giménez-Lirola Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 Software Bio-Protocol |
| title | Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 Software |
| title_full | Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 Software |
| title_fullStr | Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 Software |
| title_full_unstemmed | Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 Software |
| title_short | Proliferation Assay Using Cryopreserved Porcine Peripheral Mononuclear Cells Stimulated With Concanavalin A and Analyzed With FCS ExpressTM 7.18 Software |
| title_sort | proliferation assay using cryopreserved porcine peripheral mononuclear cells stimulated with concanavalin a and analyzed with fcs expresstm 7 18 software |
| url | https://bio-protocol.org/en/bpdetail?id=5332&type=0 |
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