A Modified Protocol for Isolation and Mitogenic Stimulation of Rat Peripheral Blood Mononuclear Cells

Document Type : Research Articles

Authors

Saeedeh Pourhamedi ¹

Maryam M. Matin ^{1, 2}

Azadeh Haghighitalab ^{1, 3, 4}

Ahmad Reza Bahrami ^{1, 2}

¹ Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran

² Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

³ Stem Cells and Regenerative Medicine Department, Academic Center for Education, Culture, and Research (ACECR) – Khorasan Razavi, Iran

⁴ Current Affiliation: Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

10.22067/jcmr.2025.96193.1123

Abstract

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 the 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 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 1.25–5 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 protocols for isolation and stimulation of rPBMCs presented here, providing a reliable framework for future in vitro studies on immune function and immunomodulation in rat models.

Keywords

Rat peripheral blood mononuclear cells

Ficoll density gradient protocol

Concanavalin A

Phytohemagglutinin

Cell viability

TNF-α secretion

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References

Bojar D., Meche L., Meng G., Eng W., Smith D. F., Cummings R. D., et al. (2022) A useful guide to lectin binding: machine-learning directed annotation of 57 unique lectin specificities. ACS Chemical Biology 17(11): 2993-3012.

Boyum A. (1968) Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g. Scandinavian Journal of Clinical Laboratory Investigation 21(Suppl 97): 77-89.

Fasanmade A. A., and Jusko W. J. (1995) Optimizing whole blood lymphocyte proliferation in the rat. Journal of Immunological Methods 184(2): 163-167.

Gholamnezhad Z., Rafatpanah H., Sadeghnia H. R., and Boskabady M. H. (2015) Immunomodulatory and cytotoxic effects of Nigella sativa and thymoquinone on rat splenocytes. Food and Chemical Toxicology 86: 72-80.

Healthcare G. (2010) Isolation of mononuclear cells: Methodology and applications.

Jiao J., Zhao X., Hou R., Wang Y., Chang W., Liang N., et al. (2019) Comparison of two commonly used methods for stimulating T cells. Biotechnology Letters 41(12): 1361-1371.

Kleiveland C. R. (2015) Peripheral blood mononuclear cells. The Impact of Food Bioactives on Health. Switzerland. Springer International Publishing (pp. 161-167).

Kohn D. F., and Barthold S. W. (2013) Biology and diseases of rats. Laboratory Animal Medicine. USA. Elsevier Academic Press (pp. 91-122).

Lijnen P., Saavedra A., and Petrov V. (1997) In vitro proliferative response of human peripheral blood mononuclear cells to concanavalin A. Clinica Chimica Acta 264(1): 91-101.

Mizobe F., Martial E., Colby-Germinario S., and Livett B. G. (1982) An improved technique for the isolation of lymphocytes from small volumes of peripheral mouse blood. Journal of Immunological Methods 48(3): 269-279.

Molaee N., Mosayebi G., Pishdadian A., Ejtehadifar M., and Ganji A. (2017) Evaluating the proliferation of human peripheral blood mononuclear cells using MTT assay. International Journal of Basic Sciences in Medicine 2(1): 25-28.

Norian R., Delirezh N., and Azadmehr A. (2015) Evaluation of proliferation and cytokines production by mitogen-stimulated bovine peripheral blood mononuclear cells. Veterinary Research Forum 6(4): 265-271.

O'flynn K., Russul-Saib M., Ando I., Wallace D. L., Beverley P., Boylston A., et al. (1986) Different pathways of human T-cell activation revealed by PHA-P and PHA-M. Immunology 57(1): 55.

Otto G. M., Franklin C. L., and Clifford C. B. (2015) Biology and diseases of rats. Laboratory Animal Medicine. USA. Elsevier Academic Press (pp. 151-207).

Pandit H., Thakur G., Gopalakrishnan A. R. K., Dodagatta-Marri E., Patil A., Kishore U., et al. (2016) Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells. Immunobiology 221(2): 310-322.

Song T., Chen M., Wang X., Zhu E., Xue Y., Wang J., et al. (2021) Intermittent hypoxia: friend or foe on endothelial repair in mouse model. Experimental Lung Research 47(5): 211-225.

Torres Crigna A., Uhlig S., Elvers-Hornung S., Klüter H., and Bieback K. (2020) Human adipose tissue-derived stromal cells suppress human, but not murine lymphocyte proliferation, via indoleamine 2, 3-dioxygenase activity. Cells 9(11): 2419.

Walter S., Jung T., Herpich C., Norman K., Pivovarova-Ramich O., and Ott, C. (2023) Determination of the autophagic flux in murine and human peripheral blood mononuclear cells. Frontiers in Cell and Developmental Biology 11: 1122998.

Wattrang E., Palm A. K., and Wagner B. (2012) Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells. Veterinary Immunology and Immunopathology 146(2): 113-124.