Culturing of Activated T Cells from Human Peripheral Blood Mononuclear Cells

Protocolo

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 g at RT.
   5. Discard the supernatant and wash the cells again by resuspending in 10 mL of RPMI 1640 and centrifuge as described in 1.1.4.
   6. Discard the supernatant and resuspend the cells at 2 x 10⁶ cells per mL of X-VIVO 15 medium + 5% human serum (hereafter: T cell medium).
   7. Plate 2 mL of the cells per well in a 24-well cell culture plate and incubate overnight at 37 °C and 5% CO₂.
2. Activation of cells (Day 0)
   1. Wash CD3/CD28 beads by transferring 12.5 µL of beads per 1 million cells to a microcentrifuge tube. Add 12.5 µL of PBS per 12.5 µL of beads.
      NOTE: It is important to vortex the vial of beads before use.
   2. Place the microcentrifuge tube on a suitable magnet for 1 min.
   3. Discard the buffer and resuspend the beads in the original volume of the T cell medium (12.5 µL of T cell medium per 12.5 µL of the original volume of beads).
   4. Add 12.5 µL of beads per million of cells corresponding to a ratio of 1:2 (beads: cells).
   5. Divide the cells into two conditions with around 5 million cells in each.
   6. Add the correct volume of cytokines to the conditions as mentioned in Table 1.
   7. Incubate the cells for 3 days at 37 °C and 5% CO₂.
3. Culturing of cells (Days 3 and 5)
   1. Resuspend the cells and split them by transferring half the volume from each well into a new well. Add the same volume of fresh T cell medium to each well.
   2. Add new cytokines to each condition as mentioned in Table 1.

	Cytokine	Stock	Dilution factor	Final
Condition 1	IL-2	3 x 106 U/mL	30,000	100 U/mL
Condition 2	IL-15	2 x 105 U/mL	2,000	100 U/mL

Table 1: Preparation of cytokine cultures used to guide metabolic changes in T cells.

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Materiales

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