Abstract
Peripheral blood is the most common source of T-lymphocytes for in vitro culture. Here, we present a simple and standardized method for small- or large-scale isolation of viable T-lymphocytes and other mononuclear cells from fresh peripheral blood or buffy coat blood samples using the density gradient centrifugation. T-cells obtained using the protocol described here can be used for a variety of downstream analysis, including cellular, molecular, and functional assays.
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References
Bøyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 21(Suppl 97):77–89
Bignold LP, Ferrante A (1987) Mechanism of separation of polymorphonuclear leukocytes from whole blood by the one-step Hypaque-Ficoll method. J Immunol Methods 96:29–33
de Almeida MC, Silva AC, Barral A, Barral Netto M (2000) A simple method for human peripheral blood monocyte isolation. Mem Inst Oswaldo Cruz 95:221–222
Dainiak MB, Kumar A, Galaev IY, Mattiasson B (2007) Methods in cell separations. Adv Biochem Eng Biotechnol 106:1–18
Fazil MHUT, Ong ST, Chalasani MLS, Low JH, Kizhakeyil A, Mamidi A, Lim CFH, Wright GW, Lakshminarayanan R, Kelleher D, Verma NK (2016) GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells. Sci Rep 6:37721
Verma NK, Fazil MHUT, Ong ST, Chalasani MLS, Low JH, Kottaiswamy A, Praseetha P, Kizhakeyil A, Kumar S, Panda AK, Freeley M, Smith SM, Boehm BO, Kelleher D (2016) LFA-1/ICAM-1 ligation in human T-cells promotes Th1 polarization through a GSK3β signalling-dependent Notch pathway. J Immunol 197:108–118
Ong ST, Freeley M, Skubis-Zegadło J, Fazil MHUT, Kelleher D, Fresser F, Baier G, Verma NK, Long A (2014) Phosphorylation of Rab5a by protein kinase Cε is crucial for T-cell migration. J Biol Chem 289:19420–19434
Verma NK, Dempsey E, Conroy J, Olwell P, Mcelligott AM, Davies AM, Kelleher D, Butini S, Campiani G, Williams DC, Zisterer DM, Lawler M, Volkov Y (2008) A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin. J Mol Med (Berl) 86:457–469
Kaplan J, Nolan D, Ree A (1982) Altered lymphocyte markers and blastogenic responses associated with 24 hour delay in processing of blood samples. J Immunol Methods 50:187–191
Grievink HW, Luisman T, Kluft C, Moerland M, Malone KE (2016) Comparison of three isolation techniques for human peripheral blood mononuclear cells: cell recovery and viability, population composition, and cell functionality. Biopreserv Biobank 14:410–415
Acknowledgments
This work was supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant to N.K.V. and the Singapore Ministry of Education under its Singapore Ministry of Education (MOE) Academic Research Fund (AcRF) Tier 1 (2014-T1-001-141) and MOE-AcRF Tier 2 (MOE2017-T2-2-004). A.K. acknowledges Ph.D. fellowship provided by Lee Kong Chian School of Medicine, Nanyang Technological University Singapore.
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Kizhakeyil, A., Ong, S.T., Fazil, M.H.U.T., Chalasani, M.L.S., Prasannan, P., Verma, N.K. (2019). Isolation of Human Peripheral Blood T-Lymphocytes. In: Verma, N. (eds) T-Cell Motility. Methods in Molecular Biology, vol 1930. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9036-8_2
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DOI: https://doi.org/10.1007/978-1-4939-9036-8_2
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