Culturing of Activated T Cells from Human Peripheral Blood Mononuclear Cells

Abstract

Source: Mølgaard, K., et al. Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells. J. Vis. Exp. (2021).

In this video, we describe a protocol for human primary T cell activation. The incubation of naïve T cells with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, followed by interleukin-2 or interleukin-15 treatment, leads to the activation of naïve T cells and their differentiation and proliferation into effector or memory T cells.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Culturing of activated human primary T lymphocytes

1. Thawing of cells (Day 1)
   1. Pre-warm 10 mL of RPMI 1640 per sample to around 37 °C.
   2. Take the desired number of peripheral blood mononuclear cell ampules and store them temporarily on dry ice.
   3. Resuspend the frozen cells in 10 mL of pre-warmed RPMI 1640.
   4. Centrifuge the cells for 5 min at 500 x

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well tissue culture plate	Nunc	142485
Anti-CD3xCD28 beads	Gibco	11161D
X-VIVO 15	Lonza	BE02-060F
Human Serum	Sigma Aldrich	H4522	Heat inactivated at 56 °C for 30 min
IL-15	Peprotech	200-02
IL-2	Peprotech	200-15
Lymphoprep	Stemcell Technologies	7801
PBS	Thermo Fisher	10010023
RPMI 1640	Gibco-Thermo Fisher	61870036
T cell beads magnet DynaMag-2 Magnet	Thermo Fisher	12321D