An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation
Published: March 29, 2024
Abstract
Source: Hsu, P., et al. Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs). J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Density Gradient Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) Add 25 ml heparinized whole blood into a 50 ml tube by a 25 ml pipette. Dilute cells with 25 ml of phosphate-buffered saline (PBS). Add 15 ml of Ficoll-Paque density gradient into a new 50 ml tube, and while tilting …
開示
The authors have nothing to disclose.
Materials
| Ficoll-Paque PLUS | GE Healthcare | 71-7167-00 AG | Density grandient for isolation of peripheral blood mononuclear cells (PBMCs) |
| Vibrant CFDA-SE Cell Tracer Kit (CFSE) | Life Technologies | V12883 | Cellular label for detection of cell division |
| Dynabeads Human T-Activator CD3/28 | Life Technologies | 111.32D | Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc. |
| autoMACS™ Separator | Miltenyi Biotec | autoMACS™ Separator | Magnetic based cell separator |
| autoMACS® Columns | Miltenyi Biotec | 130-021-101 | separation columns |
| CD14 microbeads, human | Miltenyi Biotec | 130-050-201 | For positive selection of CD14+ human monocytes and macrophages from PBMCs |
| CD4 microbeads, human | Miltenyi Biotec | 130-045-101 | For positive selection of CD4+ human T lymphocytes from PBMCs |
| RPMI 1640 Medium | Life Technologies | 11875 | Human PBMC/leukocyte culture medium |
| DMEM, Low glucose, pyruvate | Life Technologies | 11885 | Human mesenchymal stem cell (MSC) culture medium |
| L-glutamine | Life Technologies | 25030-081 | Supplementation for MSC complete medium |
| Penicillin/Streptomycin | Life Technologies | 15070-063 | Supplementation for PBMC/leukocyte and MSC complete medium |
| Fetal bovine serum (FBS) | 1) Hyclone, for MSC culture, 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations) | 1) SH30070.03M 2) 10091-148 | Pre-test lots for support of MSC in vitro culture |
| 24-well cell culture plate | Corning | COR3524 | Co-culture plate |
| 50 mL centrifuge tube | Corning | 430291 | Isolation PBMCs from whole blood by Ficoll-Paque PLUS |
| 15 mL centrifuge tube | Corning | 430766 | Collection of the labeled and unlabeled cell fractions |
| Round-bottom tubes | BD Falcon | 352008 | Collection of cells for flow cytometric analysis |
