A Modified Protocol for Isolation and Mitogen Stimulation of Rat Peripheral Blood Mononuclear Cells (rPBMCs)

Journal of Cell and Molecular Research

مقالات آماده انتشار، پذیرفته شده، انتشار آنلاین از تاریخ 01 آذر 1404

نوع مقاله: Research Articles

شناسه دیجیتال (DOI): 10.22067/jcmr.2025.96193.1123

نویسندگان

Ahmad Reza Bahrami^{* 1}؛ saeedeh pourhamedi²؛ Maryam Matin³؛ Azadeh Haghighitalab⁴

¹Ferdowsi University of Mashhad

²Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

³1 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran. 2 Novel Diagnostics and Therapeutics Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.

⁴Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.

چکیده

Peripheral blood mononuclear cells (PBMCs) are key components of the immune system and serve as a practical in vitro model in many research programs. Despite these widespread applications, popular standard methods for isolating and stimulating PBMCs in rats (rPBMCs) have remained limited. In this study, rPBMCs were isolated from rat peripheral blood using a modified Ficoll density gradient protocol, followed by a short-term adherence step to remove monocytes. A complete blood count (CBC) analysis and Giemsa staining confirmed that the isolated population consisted of approximately 98% lymphocytes, indicating high purity of the isolated cells. The cells were subsequently exposed to the different concentrations of mitogens, Concanavalin A (ConA; 1.25–10 µg/mL) and Phytohemagglutinin (PHA; 1–2%) for 3 and 5 days. Cell proliferation and viability were assessed by MTT assay, and TNF-α secretion was quantified by ELISA. ConA treatment resulted in a concentration-dependent increase in rPBMCs proliferation, with the highest viability rate observed at 1.2–5 µg/mL on day 3. At a concentration of 5 μg/mL, ConA significantly enhanced rPBMCs viability. This elevated level remained constant between days 3 and 5, indicating a sustained cellular response. By comparison, the stimulatory effect of PHA proved either weaker or delayed, as it reached its peak only on day 5. Furthermore, TNF-α analysis revealed that ConA markedly enhanced cytokine secretion, whereas PHA triggered only a modest increase. These findings demonstrate that ConA is a more potent mitogen than PHA in rat PBMCs, promoting both proliferation and cytokine release in a dose-dependent manner. The optimized ways of rPBMCs isolation and stimulation are presented here, which could provide a reliable framework for future in vitro studies of immune function and immunomodulation in rat models.

کلیدواژه‌ها

Rat peripheral blood mononuclear cells (rPBMCs)؛ Ficoll density gradient protocol؛ Concanavalin A (ConA)؛ Phytohemagglutinin (PHA)؛ Cell viability؛ TNF-α secretion