PBMCs were thawed, labeled with CTV and rested overnight. The next day, phenotypically naïve IFNγ producing cells (TVN ) and nonproducing (TN ) CD8+ T cells were isolated using an IFNγ secretion assay cell enrichment and detection kit (Miltenyi Biotec) and cell sorting. Briefly, cells were transferred onto CD3 coated plates (10 μg/mL; in the presence of soluble CD28 at 5 μg/mL), stimulated for 3 h at 37°C, labeled with IFNγ specific capture antibody reagent and incubated for 45 min at 37°C (while slowly rotating). Next, the cells were concurrently incubated with IFNγ detection antibody and antibodies against the remaining phenotypic T cell markers, which allowed for identification of naive T cell subset. IFNγ producing and nonproducing naïve CD8+ T cell subsets were isolated by cell sorting and incubated in the presence of CD3/CD2/CD28 coated beads (T cell activation/expansion kit, Miltenyi Biotec) at ratio 1:1 in the presence of IL-2 (100 U/mL) for 3 days, when proliferation and phenotype were evaluated by flow cytometry.
Ifnγ secretion assay cell enrichment and detection kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The IFNγ secretion assay cell enrichment and detection kit is a laboratory product designed to isolate and detect cells that secrete the cytokine interferon-gamma (IFNγ). The kit provides the necessary reagents and tools to enrich and identify IFNγ-producing cells from a mixed cell population.
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9 protocols using ifnγ secretion assay cell enrichment and detection kit
1
Isolation and Activation of Naïve CD8+ T Cells
2
Activation and IFN-γ Secretion Assay
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PBMCs from three healthy HLA-A02:01 positive subjects were cultured in RPMI 1640 (Gibco) supplemented with 2 mM L-Glut (HyClone), 10% human serum (Sigma-Aldrich), 100 IU/ml penicillin and 100 μg/ml streptomycin (Capricorn). Cells were maintained at 37 °C in a humidified incubator with 5% CO2. PBMCs were seeded at 2.5 × 106 cells/ml in 3 ml in a 6 well plate and cultured in presence of IL-2 (Sigma) at a final concentration of 10 U/mL and 25 µL/mL of ImmunoCult™ Human CD3/CD28 T Cell Activator (StemCell technologies). Following 3 days incubation, the interferon-gamma (IFN-γ) production was evaluated through the IFN-γ Secretion Assay –Cell Enrichment and Detection Kit (Miltenyi Biotec). Briefly, cells were harvested, centrifuged and incubated 4 h at 37 °C with SSX2, SSX2-BACT2 and SSX-BACT3 peptides at a final concentration of 10 uM. Unstimulated and PHA stimulated PBMCs were used, respectively, as negative and positive controls. Subsequently, cells were washed and stained with IFN-γ Catch Reagent, incubated 45 min at 37 °C, centrifuged and labelled with IFN-γ Detection Antibody (PE), CD8 PE-Cy7 (Life Technologies) and CD3 super bright 436 (Invitrogen). After 15 min incubation on ice, cell were washed, resuspended in 500uL of cold buffer and analysed by flow cytometry (AttuneNxT-LifeTechnologies).
3
Isolation and Activation of Naïve CD8+ T Cells
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PBMCs were thawed, labeled with CTV and rested overnight. The next day, phenotypically naïve IFNγ producing cells (TVN ) and nonproducing (TN ) CD8+ T cells were isolated using an IFNγ secretion assay cell enrichment and detection kit (Miltenyi Biotec) and cell sorting. Briefly, cells were transferred onto CD3 coated plates (10 μg/mL; in the presence of soluble CD28 at 5 μg/mL), stimulated for 3 h at 37°C, labeled with IFNγ specific capture antibody reagent and incubated for 45 min at 37°C (while slowly rotating). Next, the cells were concurrently incubated with IFNγ detection antibody and antibodies against the remaining phenotypic T cell markers, which allowed for identification of naive T cell subset. IFNγ producing and nonproducing naïve CD8+ T cell subsets were isolated by cell sorting and incubated in the presence of CD3/CD2/CD28 coated beads (T cell activation/expansion kit, Miltenyi Biotec) at ratio 1:1 in the presence of IL-2 (100 U/mL) for 3 days, when proliferation and phenotype were evaluated by flow cytometry.
4
IFN-γ-Secreting CD4+ T Cell Isolation
5
Isolation and Culture of SARS-CoV-2-Specific T Cells
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Virus-specific T cells were isolated from PBMCs derived from 100 mL of peripheral blood from convalescent donors following a 6-h stimulation with overlapping SARS-CoV-2-specific peptide pools (JPT Peptide Technologies; 1 μg/mL each) using an IFN-γ Secretion Assay—Cell Enrichment and Detection Kit according to the manufacturer’s instructions (Miltenyi Biotec). Isolated virus-specific T cells were cultured in complete medium (VLE RPMI 1640 [PAN-Biotech] supplemented with penicillin [100 IU/mL], streptomycin [Biochrom], 10% fetal calf serum [FCS, PAA], 10 ng/mL recombinant human IL-7 [rhIL-7] and rhIL-15 [CellGenix]) in 24-well plates, in humidified incubators at 37°C and 5% CO2 as described previously.88 (link),122 (link) Cells were split 1:1 upon reaching 100% confluency.
6
Enrichment of COVID-19 IFN-γ T Cells
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For the enrichment of IFN-γ-secreting T cells, peptide pools (5 μM/peptide) for 6 h were used to stimulate the PBMCs of recovered COVID-19 patients. Then, IFN-γ-secreting T cells were caught using IFN-γ Secretion Assay Cell Enrichment and Detection Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+T cells were selected by EasySep Human CD4+ T Cell Enrichment Kit (Stemcell).
7
Enrichment of WT1-specific T cells
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An IFN-γ CSA (IFN-γ Secretion Assay–Cell Enrichment and Detection Kit, Miltenyi Biotec) was used to evaluate the effect of SnMP on the enrichment of WT1-specific T cells. Freshly isolated PBMCs were resuspended at a concentration of 1 × 107 cells/mL in TexMACS GMP Medium (Miltenyi Biotec) and allowed to rest overnight. WT1-specific T cells were enriched by using the CSA to mimic a situation very comparable to a clinical setting. PBMCs were stimulated with ppWT1 in the presence and absence of SnMP overnight. This was followed by the detection of IFN-γ secreting cells and their enrichment according to the manufacturer’s instructions.
For flow cytometric analysis, cells were stained before and after enrichment with mAbs against CD3, CD8, CD56 and CD45. Dead cells were excluded by using 7-amino-actinomycin D (7-AAD). Gates were based on the light scatter properties of lymphocytes and on CD3+/IFN-γ+ cell populations. The PE-conjugated anti-IFN-γ antibody was obtained from Miltenyi Biotec.
For differentiation between antiviral and WT1-specific T-cell responses, PBMCs were stimulated overnight with pools of EBV-derived (1 µg/mL pro peptide) and WT1-derived peptides with or without SnMP. The IFN-γ CSA was performed as described.
For flow cytometric analysis, cells were stained before and after enrichment with mAbs against CD3, CD8, CD56 and CD45. Dead cells were excluded by using 7-amino-actinomycin D (7-AAD). Gates were based on the light scatter properties of lymphocytes and on CD3+/IFN-γ+ cell populations. The PE-conjugated anti-IFN-γ antibody was obtained from Miltenyi Biotec.
For differentiation between antiviral and WT1-specific T-cell responses, PBMCs were stimulated overnight with pools of EBV-derived (1 µg/mL pro peptide) and WT1-derived peptides with or without SnMP. The IFN-γ CSA was performed as described.
8
Isolating HPV 16 E6-specific CD4 T-cells
9
Multiparameter Flow Cytometry Assay
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