Comparison of three monocyte depletion techniques for lymphocyte isolation from peripheral blood mononuclear cell

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Passaworn Cheyasawan
Nuchjira Takheaw
Supansa Pata
Watchara Kasinrerk
Kantinan Chuensirikulchai
Witida Laopajon


Background: Lymphocytes are crucial cells in the immune system. Studying lymphocyte function can lead to better understanding of the immune system. Essentially, lymphocyte isolation technique is required for studying lymphocyte function. Several techniques were developed to prepare lymphocytes, including depletion of monocytes from peripheral blood mononuclear cells (PBMCs).

Objectives: To compare utilization of three different techniques for lymphocyte isolation by monocyte depletion from PBMCs.

Materials and methods: Lymphocytes were isolated from PBMCs by depletion of monocytes using (i) magnetic beads phagocytosis, (ii) Percoll density gradient centrifugation, and (iii) anti-FITC antibody conjugated micro-magnetic beads. The number of cells collected was counted using Turk’s solution. The cellular profiles of PBMCs and monocyte-depleted PBMCs were determined by immunofluorescence and flow cytometry.

Results: The highest yield and purity of monocyte-depleted PBMCs were achieved using the anti-FITC antibody conjugated microbeads depletion method. However, this method consumed the longest time and had the highest cost. Magnetic beads phagocytosis depletion method required the shortest time; however, the wide range of collected yield was a concern. Percoll gradient centrifugation method was the cheapest, but the percentage yield was the lowest among the three methods.

Conclusion: The utilization of three different methods was able to deplete monocytes from PBMCs. However, each technique had some advantages and disadvantages. The information obtained from this study might give some guidance for selecting a suitable method for isolation of lymphocytes based on the monocyte-depleted PBMC strategy.


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Cheyasawan, P., Takheaw, N., Pata, S., Kasinrerk, W., Chuensirikulchai, K., & Laopajon, W. (2021). Comparison of three monocyte depletion techniques for lymphocyte isolation from peripheral blood mononuclear cell. Journal of Associated Medical Sciences, 55(1), 24–31. Retrieved from
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Abbas AK, Lichtman AH, Pillai S, Abbas AK. Cellular and molecular immunology. 8th Ed. Phildelphia: Elsevier Saunders; 2014.

Abbas AK, Lichtman AH, Pillai S, Abbas AK. Cellular and molecular immunology. 9th Ed. Phildelphia: Elsevier Saunders; 2018.

Milioglou I, Kalaitzidou I, Ladomenou F. Interpretation of lymphocyte subset counts by the general pediatrician. Pediatr Int. 2019; 61(1): 16-22.

Adler LN, Jiang W, Bhamidipati K, Millican M, Macaubas C, Hung S-c, et al. The Other Function: Class II-Restricted Antigen Presentation by B Cells. 2017; 8(319).

Hong S, Zhang Z, Liu H, Tian M, Zhu X, Zhang Z, et al. B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4(+) T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen. Immunity. 2018; 49(4): 695-708.

Swain SL, McKinstry KK, Strutt TM. Expanding roles for CD4(+) T cells in immunity to viruses. Nat Rev Immunol. 2012; 12(2): 136-48.

Vidal SM, Khakoo SI, Biron CA. Natural killer cell responses during viral infections: flexibility and conditioning of innate immunity by experience. Curr Opin Virol. 2011; 1(6): 497-512.

Blumenreich MS. The White Blood Cell and Differential Count. In: rd, Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. Boston; 1990.

Chiu P-L, Chang C-H, Lin Y-L, Tsou P-H, Li B-R. Rapid and Safe Isolation of Human Peripheral Blood B and T Lymphocytes through Spiral Microfluidic Channels. Scientific Reports. 2019; 9(1): 8145.

Mohr F, Przibilla S, Leonhardt F, Stemberger C, Dreher S, Müller TR, et al. Efficient immunoaffinity chromatography of lymphocytes directly from whole blood. Scientific Reports. 2018; 8(1): 16731.

Plouffe BD, Murthy SK, Lewis LH. Fundamentals and application of magnetic particles in cell isolation and enrichment: a review. Rep Prog Phys. 2015; 78(1): 016601.

Chiu PL, Chang CH, Lin YL, Tsou PH, Li BR. Rapid and Safe Isolation of Human Peripheral Blood B and T Lymphocytes through Spiral Microfluidic Channels. Sci Rep. 2019; 9(1): 8145.

Rahmanian N, Bozorgmehr M, Torabi M, Akbari A, Zarnani AH. Cell separation: Potentials and pitfalls. Prep Biochem Biotechnol. 2017; 47(1): 38-51.

Weinstock A, Fisher EA. Methods to Study Monocyte and Macrophage Trafficking in Atherosclerosis Progression and Resolution. Methods Mol Biol. 2019; 1951: 153-65.

Fluks AJ. Three-step isolation of human blood monocytes using discontinuous density gradients of Percoll. J Immunol Methods. 1981; 41(2): 225-33.

de Almeida MC, Silva AC, Barral A, Barral Netto M. A simple method for human peripheral blood monocyte isolation. Mem Inst Oswaldo Cruz. 2000; 95(2): 221-3.

Huhn M, Juan MHS, Melcher B, Dreis C, Schmidt KG, Schwiebs A, et al. Inflammation-Induced Mucosal KYNU Expression Identifies Human Ileal Crohn's Disease. Journal of clinical medicine. 2020; 9(5): 1360.

Fuss IJ, Kanof ME, Smith PD, Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol. 2009; 7(7): 1.

Chen H, Schurch CM, Noble K, Kim K, Krutzik PO, O'Donnell E, et al. Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes. BMC Immunol. 2020; 21(1): 15.

Repnik U, Knezevic M, Jeras M. Simple and cost-effective isolation of monocytes from buffy coats. J Immunol Methods. 2003; 278(1-2): 283-92.

Biswas P, Mantelli B, Sica A, Malnati M, Panzeri C, Saccani A, et al. Expression of CD4 on human peripheral blood neutrophils. Blood. 2003; 101(11): 4452-6.

Sabroe I, Jones EC, Usher LR, Whyte MK, Dower SK. Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. J Immunol. 2002; 168(9): 4701-10.

Suzuki T, Hashimoto S, Toyoda N, Nagai S, Yamazaki N, Dong HY, et al. Comprehensive gene expression profile of LPS-stimulated human monocytes by SAGE. Blood. 2000; 96(7): 2584-91.

Burger D, Dayer JM. The role of human T-lymphocyte-monocyte contact in inflammation and tissue destruction. Arthritis Res. 2002; 4(3): 169-76.

Takheaw N, Laopajon W, Surinkaew S, Khummuang S, Pata S, Kasinrerk W. Ligation of Na, K ATPase beta3 subunit on monocytes by a specific monoclonal antibody mediates T cell hypofunction. PLoS One. 2018; 13(6): e0199717.

Powell DJ, Jr., Brennan AL, Zheng Z, Huynh H, Cotte J, Levine BL. Efficient clinical-scale enrichment of lymphocytes for use in adoptive immunotherapy using a modified counterflow centrifugal elutriation program. Cytotherapy. 2009; 11(7): 923-35.

Mizobe F, Martial E, Colby-Germinario S, Livett BG. An improved technique for the isolation of lymphocytes from small volumes of peripheral mouse blood. J Immunol Methods. 1982; 48(3): 269-79.

Gu BJ, Sun C, Fuller S, Skarratt KK, Petrou S, Wiley JS. A quantitative method for measuring innate phagocytosis by human monocytes using real-time flow cytometry. Cytometry A. 2014; 85(4): 313-21.

ZHANG R, INAGAWA H, TAKAHASHI M, KAWANISHI H, KAZUMURA K, TSUCHIYA H, et al. Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis. 2017; 37(7): 3897-903.

Martinez-Riano A, Bovolenta ER, Mendoza P, Oeste CL, Martin-Bermejo MJ, Bovolenta P, et al. Antigen phagocytosis by B cells is required for a potent humoral response. EMBO Rep. 2018; 19(9).

Holtrop S, Rijke-Schilder GP, Tamboer WP, Koene RA, Tax WJ. Removal of monocytes from cell suspensions with anti-CD14 antibody and carbonyl-iron, using Fc gamma R-dependent accessory function as a sensitive measure of monocyte presence. J Immunol Methods. 1992; 156(2): 217-22.

Feucht HE, Hadam MR, Frank F, Riethmuller G. Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (percoll). J Immunol Methods. 1980; 38(1-2): 43-51.

Harbeck RJ, Hoffman AA, Redecker S, Biundo T, Kurnick J. The isolation and functional activity of polymorphonuclear leukocytes and lymphocytes separated from whole blood on a single percoll density gradient. Clin Immunol Immunopathol. 1982; 23(3): 682-90.

Horgan K, Shaw S, Boirivant M. Immunomagnetic purification of T cell subpopulations. Curr Protoc Immunol. 2009; 7(7): 4.

Moonen CGJ, Karlis GD, Schoenmaker T, Forouzanfar T, Loos BG, de Vries TJ. T Cell Proliferation Is Induced by Chronically TLR2-Stimulated Gingival Fibroblasts or Monocytes. 2019; 20(24): 6134.