Isolation of Human Peripheral Blood T-Lymphocytes

  • Atish KizhakeyilEmail author
  • Seow Theng Ong
  • Mobashar Hussain Urf Turabe Fazil
  • Madhavi Latha Somaraju Chalasani
  • Praseetha Prasannan
  • Navin Kumar Verma
Part of the Methods in Molecular Biology book series (MIMB, volume 1930)


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.

Key words

T-Lymphocytes PBMC Density gradient Ficoll 



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.


  1. 1.
    Bøyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 21(Suppl 97):77–89Google Scholar
  2. 2.
    Bignold LP, Ferrante A (1987) Mechanism of separation of polymorphonuclear leukocytes from whole blood by the one-step Hypaque-Ficoll method. J Immunol Methods 96:29–33CrossRefGoogle Scholar
  3. 3.
    de Almeida MC, Silva AC, Barral A, Barral Netto M (2000) A simple method for human peripheral blood monocyte isolation. Mem Inst Oswaldo Cruz 95:221–222CrossRefGoogle Scholar
  4. 4.
    Dainiak MB, Kumar A, Galaev IY, Mattiasson B (2007) Methods in cell separations. Adv Biochem Eng Biotechnol 106:1–18PubMedGoogle Scholar
  5. 5.
    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 6:37721CrossRefGoogle Scholar
  6. 6.
    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 197:108–118CrossRefGoogle Scholar
  7. 7.
    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 289:19420–19434CrossRefGoogle Scholar
  8. 8.
    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) 86:457–469CrossRefGoogle Scholar
  9. 9.
    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 50:187–191CrossRefGoogle Scholar
  10. 10.
    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 14:410–415CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Atish Kizhakeyil
    • 1
    Email author
  • Seow Theng Ong
    • 1
  • Mobashar Hussain Urf Turabe Fazil
    • 1
  • Madhavi Latha Somaraju Chalasani
    • 2
    • 1
  • Praseetha Prasannan
    • 1
  • Navin Kumar Verma
    • 3
  1. 1.Lymphocyte Signalling Research Laboratory, Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore
  2. 2.Autoimmunity and Inflammation ProgramHospital for Special SurgeryNew YorkUSA
  3. 3.Lee Kong Chian School of MedicineNanyang Technological University SingaporeSingaporeSingapore

Personalised recommendations