In this application protocol, pan T cells are isolated directly from human PBMCs by magnetic enrichment using the Pan T Cell Isolation Kit, human and subsequently activated and expanded with the T cell Activation and Expansion Kit, human. T cell purity, proliferation, and expression of activation markers are assessed by flow cytometry at different time points.
PBS-EDTA: Phosphate-buffered saline (PBS), pH 7.2, with 2 mM EDTA.
▲Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
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Freshly prepare the following master mix for each sample:
10 µL of each antibody:
CD3-FITC
CD2-PE
Store master mix in the dark in the refrigerator (2–8 °C) until use. Do not store for extended periods.
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Aliquots of the stock solution should be stored at –20 °C or below. Avoid repeated freeze-thaw cycles.
Prepare TexMACS Medium supplemented with human IL-2 (50 IU/mL).
▲ Note: Make sure to add IL-2 freshly to the T cell medium for cell expansion.
▲ Note: Instead of TexMACS Medium, RPMI supplemented with FBS (10 % final concentration), 100× L-glutamine stock solution (1% final concentration), 2-mercaptoethanol (0.01 mM final concentration) and human IL-2 (50 IU/mL) can be used for T cell cultivation and expansion.
▲ Note: Addition of penicillin/streptomycin to the T cell medium is optional (e.g., 100× penicillin/streptomycin stock solution to a final concentration of 1%).
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Prepare TexMACS™ Medium supplemented with human IL-2 (50 IU/mL).
▲ Note: Make sure to add IL-2 freshly to the T cell medium for cell expansion.
▲ Note: Instead of TexMACS Medium, RPMI supplemented with FBS (10% final concentration), 100× L-glutamine stock solution (1% final concentration), 2-mercaptoethanol (0.01 mM final concentration) and human IL-2 (50 IU/mL) can be used for T cell cultivation and expansion.
▲ Note: Addition of penicillin/streptomycin to the T cell medium is optional (e.g., 100× penicillin/streptomycin stock solution to a final concentration of 1%).
It is recommended to reconstitute lyophilized Human IL-2 IS, premium grade with deionized sterile-filtered water to a final concentration of 0.1–1.0 mg/mL in a volume of at least 100 µL.
▲ Note: Further dilutions should be prepared with 0.1% BSA or human serum albumin (HSA) in PBS.
The ED₅₀ is ≤0.2 ng/mL corresponding to a specific activity of ≥5.0×106 IU/mg (calibrated with NIBSC 86/504) or ≥1×10⁷ IU/mg (calibrated with Proleukin®).
Recommended stock concentration: 0.1mg/mL by reconstituting a 10 µg vial of Human IL-2 IS, premium grade with 100 µL deionized sterile-filtered water. This results in a final activity of 500 IU/µL.
Upon reconstitution aliquots should be stored at –20 °C or below. Avoid repeated freeze-thaw cycles.
To obtain a cell culture medium supplemented with 50 IU/mL, add 1.0 µL reconstituted Human IL-2 IS, premium grade freshly to 10 mL cell culture medium.
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Freshly prepare the following master mix for each sample:
Store master mix in the dark in the refrigerator (2–8 °C) until use. Do not store for extended periods.
▲ Note: Highly activated T cells might down-regulate CD3 to some extent. Alternatively, CD4 and CD8 stainings (e.g., with CD4-VioBlue® and CD8-VioGreen™) can be used to properly gate on T cell subpopulations.
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MACSiMAG™ Separator (#130-092-168)
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