Analyzing T Cell Phenotypes by ICS

To evaluate the phenotypes of T cells that responded to stimulation in IFN-γ ELISPOT assays, we conducted intracellular cytokine staining (ICS). The in vitro stimulation was similar to that described above in the IFN-γ ELLISPOT section. Briefly, after 12-14 days in vitro culture, PBMCs were restimulated with peptide. After 1 h, Brefeldin A was added to inhibit protein transport. After 4 additional hours of incubation, the cells were stained with T-cell surface markers CD4-PerCP (cat. 345770), CD8- FITC (cat. 345772), CD3-APC-H7 (cat. 560275) and a dead cell marker FVS510 (564406) (all from BD Biosciences). Samples were then fixed and permeabilized using eBioscience™ Fixation/Permeabilization buffers (eBioscience, cat. 00-5123-43, 00-5223-56) and stained with IFNg-APC (cat.341117, BD Biosciences), TNFa-BV421 (cat.562783, BD Biosciences) in eBioscience permeabilization buffer (eBioscience, cat. 00-8333-56) and analyzed on FACSCanto™ II (BD Biosciences) using BD FACSDiva software version 8.0.2 as described previously (32 (link)).

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Grauslund J.H., Holmström M.O., Martinenaite E., Lisle T.L., Glöckner H.J., El Fassi D., Klausen U., Mortensen R.E., Jørgensen N., Kjær L., Skov V., Svane I.M., Hasselbalch H.C, & Andersen M.H. (2023). An arginase1- and PD-L1-derived peptide-based vaccine for myeloproliferative neoplasms: A first-in-man clinical trial. Frontiers in Immunology, 14, 1117466.

Publication 2023

CD4-PerCP CD8- FITC CD3-APC-H7 FVS510 eBioscience™ Fixation/Permeabilization buffers eBioscience permeabilization buffer FACSCanto™ II

Assays Brefeldin a Buffer Cd4 t cell Cell Cytokine Elispot Fitc Ifng Inhibit Intracellular Peptide Phenotypes Protein transport T cells Tnfa

Corresponding Organization :

Other organizations : Copenhagen University Hospital, University of Copenhagen, Zealand University Hospital

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Variable analysis

independent variables

Peptide stimulation of PBMCs

dependent variables

Phenotypes of T cells that responded to stimulation
Expression of IFN-γ and TNF-α in T cells

control variables

Culture duration of 12-14 days
Addition of Brefeldin A to inhibit protein transport
Incubation duration of 4 additional hours after Brefeldin A addition
T-cell surface markers used for staining (CD4, CD8, CD3)
Dead cell marker (FVS510) used for staining
Fixation and permeabilization of samples using eBioscience buffers
Staining for IFN-γ and TNF-α in permeabilized samples
Analysis on FACSCanto II using BD FACSDiva software version 8.0.2

positive controls

None specified

negative controls

None specified

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