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Data and images for SARS-CoV-2 T Cell Analysis Kits (PBMC), human

Figures

Figure 1

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Procedure of SARS-CoV-2–reactive T cell analysis:
Peripheral blood mononuclear cells (PBMCs) are cultured with the SARS-CoV-2 PepTivator
®
Peptide Pool or control of choice (e.g. CytoStim™) for a total of 6 hours. Upon activation, cells start to upregulate activation markers and secrete cytokines. After 2 hours of cultivation, Brefeldin A is added to the cells to inhibit transport of proteins to the cellular membrane,
thereby enabling intracellular cytokine staining. After the stimulation phase cells are stained with the live/dead marker Viobility 405/452 Fixable Dye. Next, cells are fixed, permeabilized, and stained for lineage and activation markers as well as cytokines using the flow panel provided in the kit. Cells are then analyzed by flow cytometry, e.g., on a MACSQuant
®
Analyzer.

Figure 1

Procedure of SARS-CoV-2–reactive T cell analysis:
Peripheral blood mononuclear cells (PBMCs) are cultured with the SARS-CoV-2 PepTivator
®
Peptide Pool or control of choice (e.g. CytoStim™) for a total of 6 hours. Upon activation, cells start to upregulate activation markers and secrete cytokines. After 2 hours of cultivation, Brefeldin A is added to the cells to inhibit transport of proteins to the cellular membrane,
thereby enabling intracellular cytokine staining. After the stimulation phase cells are stained with the live/dead marker Viobility 405/452 Fixable Dye. Next, cells are fixed, permeabilized, and stained for lineage and activation markers as well as cytokines using the flow panel provided in the kit. Cells are then analyzed by flow cytometry, e.g., on a MACSQuant
®
Analyzer.

Figure 2

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Hierarchical gating strategy according to figure 3–6.

Figure 2

Hierarchical gating strategy according to figure 3–6.

Figure 3

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Pre-gating strategy to identify total CD3+ T cells, CD4+ T cells, and CD8+ T cells:
The following flow cytometric analysis was performed with the eight fluorochrome-conjugated antibodies and the live/dead marker that are included in the kit (see data sheet). This allows identification of SARS-CoV-2–reactive T cells in human PBMC samples obtained from convalescent or vaccinated donors. Here, isolated PBMCs of a SARS-CoV-2–reactive donor
were left untreated or stimulated with PepTivator® SARS-CoV-2 Prot_S Complete (see data sheet) and analyzed using the MACSQuant® Analyzer 16. The gating strategy allows to determine cell counts and frequencies of total CD3
+
SARS-CoV-2–reactive T cells (figure 4) and SARS-CoV-2–reactive T cell subsets (CD4
+
T cells (figure 5) and CD8
+
T cells (figure 6)). A hierarchical gating strategy is depicted in figure 2.

Figure 3

Pre-gating strategy to identify total CD3+ T cells, CD4+ T cells, and CD8+ T cells:
The following flow cytometric analysis was performed with the eight fluorochrome-conjugated antibodies and the live/dead marker that are included in the kit (see data sheet). This allows identification of SARS-CoV-2–reactive T cells in human PBMC samples obtained from convalescent or vaccinated donors. Here, isolated PBMCs of a SARS-CoV-2–reactive donor
were left untreated or stimulated with PepTivator® SARS-CoV-2 Prot_S Complete (see data sheet) and analyzed using the MACSQuant® Analyzer 16. The gating strategy allows to determine cell counts and frequencies of total CD3
+
SARS-CoV-2–reactive T cells (figure 4) and SARS-CoV-2–reactive T cell subsets (CD4
+
T cells (figure 5) and CD8
+
T cells (figure 6)). A hierarchical gating strategy is depicted in figure 2.

Figure 4

View details
Identification of total SARS-CoV-2–reactive CD3+ T cells:
Gating on total viable CD3
+
T cells (figure 3D) allows to determine cell counts and frequencies of total CD3
+
SARS-CoV-2–reactive T cells.

Figure 4

Identification of total SARS-CoV-2–reactive CD3+ T cells:
Gating on total viable CD3
+
T cells (figure 3D) allows to determine cell counts and frequencies of total CD3
+
SARS-CoV-2–reactive T cells.

Figure 5

View details
Identification of SARS-CoV-2–reactive CD4+ T cells:
Gating on total viable CD4
+
T cells (figure 3E) allows to determine SARS-CoV-2–reactive CD4
+
T cells.

Figure 5

Identification of SARS-CoV-2–reactive CD4+ T cells:
Gating on total viable CD4
+
T cells (figure 3E) allows to determine SARS-CoV-2–reactive CD4
+
T cells.

Figure 6

View details
Identification of SARS-CoV-2–reactive CD8+ T cells:
Gating on total viable CD8
+
T cells (figure 3E) allows to determine SARS-CoV-2–reactive CD8
+
T cells.

Figure 6

Identification of SARS-CoV-2–reactive CD8+ T cells:
Gating on total viable CD8
+
T cells (figure 3E) allows to determine SARS-CoV-2–reactive CD8
+
T cells.

Specifications for SARS-CoV-2 T Cell Analysis Kits (PBMC), human

Overview

The SARS-CoV-2 T Cell Analysis Kits (PBMC), human have been developed for the fast and easy detection of SARS-CoV-2–reactive T cells by intra- and extracellular staining of activation markers and cytokines. The use of 96-well cell culture plates enables the processing and analysis of multiple samples in parallel. The kits are available in different variants either containing a SARS-CoV-2 PepTivator
®
Peptide Pool of choice or not (see different kit variants). Additional SARS-CoV-2 PepTivators can be purchased separately to create individual stimulation mixtures. All kits contain antibodies for the identification of CD4
+
and CD8
+
T cells, the exclusion of monocytes and B cells as well as the staining of activation markers and cytokines. Furthermore, a positive control (CytoStim™), a live/dead marker (Viobility™ 405/452 Fixable Dye), Brefeldin A and reagents (Inside Fix and Inside Perm) for the fixation and permeabilization of cells after stimulation are included. The optimized flow panel and protocol ensure a comprehensive and efficient analysis of SARS-CoV-2–reactive T cells.
Please note: The SARS-CoV-2 T Cell Analysis Kit (PBMC), human (130-128-034) does not contain a PepTivator Peptide Pool which allows for more flexibility.

Detailed product information

Background information

T lymphocytes execute and control immunological reactions with a repertoire of cytokines, cytotoxic substances, and other mediators. The quantitative and qualitative analysis of CD4
+
T cells and CD8
+
T cells specifically recognizing and reacting towards a defined antigen provide important information to understand their function in various immunological situations. The presence of these cells indicate an infected or convalescent donor and may also allow conclusions on disease progress, severity, specific immune reaction and status
1-8
. Antigen-reactive T cells can thereby be identified and characterized by analyzing their effector function, e.g. upregulation of activation markers and production of cytokines. In the context of COVID-19 the SARS-CoV-2 T cell Analysis kit (PBMC), human, represents a sensitive and precise multiparameter assay to efficiently asses the SARS-CoV-2-reactive T cell response.

Applications

The use of 96-well cell culture plates enables the analysis of multiple samples in parallel. The kit contains a SARS-CoV-2 PepTivator
®
Peptide Pool of choice, antibodies for the identification of CD4
+
and CD8
+
T cells, the exclusion of monocytes and B cells as well as the staining of activation markers and cytokines. Furthermore a positive control (CytoStim™), a live/dead marker (Viobility™ 405/452 Fixable Dye), Brefeldin A and reagents (Inside Fix and Inside Perm) for the fixation and permeabilization of cells after stimulation are included. The optimized flow panel and protocol ensure a comprehensive and efficient analysis of SARS-CoV-2-reactive T cells in the context of:
  • Rapid detection of CD4+ and CD8+ SARS-CoV-2-reactive T cells
  • Phenotypical characterization of activated T cells after stimulation with SARS-CoV-2 PepTivator® Peptide Pools by flow cytometry
  • Immunomonitoring of SARS-CoV-2-reactive T cells
  • Research and monitoring of infection- and vaccine-specific T cell responses in individuals

Resources for SARS-CoV-2 T Cell Analysis Kits (PBMC), human

Certificates

Please follow this
link
to search for Certificates of Analysis (CoA) by lot number.

References for SARS-CoV-2 T Cell Analysis Kits (PBMC), human

Publications

  1. Swadling, L. et al. (2020) T cells in COVID-19 - united in diversity. Nat. Immunol. 21(11): 1307-1308
  2. Jarjour, N. N. et al. (2021) T Cell Memory: Understanding COVID-19. Immunity 54(1): 14-18
  3. Thieme, C. J. et al. (2020) Robust T Cell Response Toward Spike, Membrane, and Nucleocapsid SARS-CoV-2 Proteins Is Not Associated with Recovery in Critical COVID-19 Patients. Cell Rep Med. 1(6): 100092
  4. Kusnadi, A. et al. (2021)
    Severely ill COVID-19 patients display augmented functional properties in SARS-CoV-2-reactive CD8
    +
    T cells.
    Sci Immunol 6: eabe4782
  5. Meckiff, J. et al. (2020)
    Imbalance of Regulatory and Cytotoxic SARS-CoV-2-Reactive CD4
    +
    T Cells in COVID-19.
    Cell 183(5): 1340-1353
  6. Braun, J. et al. (2020) SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19. Nature 587(7833): 270-274
  7. Bacher, P. et al. (2020)
    Low avidity CD4
    +
    T cell responses to SARS-CoV-2 in unexposed individuals and humans with severe COVID-19.
    Immunity 53: 1258-1271
  8. Dan, J. et al. (2021) Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science 371: eabf4063

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