For an ELISpot or FluoroSpot assay to tell us anything, in most cases we need to stimulate the cells in vitro. Stimuli should be used for identifying antigen specific immune responses as well as for assay controls. In this feature, we’ll walk you through the optimal use of different polyclonal and antigen-specific stimuli.
Choosing the relevant compounds for stimulation is largely dependent upon the cell type and the nature of your experiment. Stimuli can be monoclonal antibodies or peptides, synthetic compounds, or substances purified from bacteria or plants.
We offer many different polyclonal and antigen-specific stimuli reagents, all validated and optimized for use in ELISpot and FluoroSpot. Most of our stimuli reagents are either monoclonal antibodies or peptide pools (we call ours PepPools). PepPools can be used as assay controls or to identify antigen specific immune responses.
Stimuli for specific research questions
When using kits from us, you might be interested in evaluating a vaccine candidate of yours, a cancer antigen, or an allergen that you’ve been interested in for a while. That’s all good and how we initially intended for our kits to be used – we supply the common material; you bring the specific stimulus.
However, we’ve seen an increasing need for high-quality and optimized stimuli that can be used for specific research questions. Enter: Our very own peptide pool portfolio, PepPool. PepPools are especially beneficial for you who work with a pathogen but lack access to or knowledge of suitable antigens. Another advantage is that you can be sure that they have been validated for ELISpot and FluoroSpot, and in individuals confirmed to have had the infection in question.
Most PepPools can also be used for flow cytometry
PepPools comes in a defined or a scanning format:
- Scanning pools do not require prior knowledge about epitopes and are designed to cover whole proteins or domains. They are often produced as 15-mers overlapping by 11 amino acids, spanning the whole protein of interest. 15-mers enable CD4+ T-cell stimulation, and, when added to cells in culture, they can be trimmed by proteases to shorter peptides, allowing for CD8+ T-cell stimulation. Thus, they can be used to stimulate any T cell and actually samples of any species. In general, scanning pools are a safer bet if you are unsure of the HLA/MHC composition in your cohort.
- PepPools with a species stated in their product name include defined peptides for specific HLA/MHC molecules. These peptides have been identified by us or others to be immunodominant and should therefore give strong immune responses. If you know that the HLA composition in your cohort is covered, then defined PepPools are the preferred choice.
We offer PepPools for a selection of common pathogens and the list is constantly growing. Just let us know if there are any peptide pools you would like to see in our portfolio. If you are looking to using our PepPools for research and don't have any ELISpot or FluoroSpot kit yet, check out our Path kits – complete and validated kits with the PepPools included.
Please note that our PepPools can be used for research questions, but as for example EBV and CMV are highly prevalent in the human population, some pools can also be used as positive controls.
|Table 1: PepPools for specific research questions|
|Stimuli||Cell type stimulated||Comment||Stimulates secretion|
|PepPool: TB (EspC), scanning||T cells (scanning pool)||T cell epitopes from the Mycobacterium tuberculosis ESX-1 secretion-associated protein EspC||IFN-γ, IL-2|
|PepPool: TB (ESAT-6), scanning||T cells (scanning pool)||T cell epitopes from the Mycobacterium tuberculosis early secretory antigenic target (ESAT-6) protein||IFN-γ, IL-2|
|PepPool: TB (CFP-10), scanning||T cells (scanning pool)||T cell epitopes from the Mycobacterium tuberculosis culture filtrate protein (CFP-10)||IFN-γ, IL-2|
|PepPool: CMV (CD4 and CD8), human||CD4+ and CD8+ T cells||T cell epitopes from Cytomegalovirus (CMV) proteins||IFN-γ, IL-2, IL-5, IL-13|
|PepPool: CyCMV+RhCMV (TP-UL83+Rh112), scanning||T cells (Macaque)||T cell epitopes from cynomolgus cytomegalovirus (CyCMV) Tegument phosphoprotein UL83, and from rhesus cytomegalovirus (rhCMV) Rh112||IFN-γ, IL-2|
|PepPool: EBV (CD4 and CD8), human||CD4+ and CD8+ T cells||T cell epitopes from latent and lytic Epstein-Barr virus (EBV) proteins.||IFN-γ, IL-2|
|PepPool: AdV5 (hexon), human||T cells||T cell epitopes from the human adenovirus 5 (hAdV5) hexon protein||IFN-γ, IL-2|
|PepPool: Vaccinia (CD4), human||CD4 T cells||T cell epitopes from several Vaccinia proteins. Of the included peptides, 91% share >90% homology with mpox virus||IFN-γ|
|PepPool: Vaccinia (CD8), human||CD8 T cells||T cell epitopes from several Vaccinia proteins. Of the included peptides, 79% share >90% homology with mpox virus||IFN-γ|
|PepPool: SARS-CoV-2 (SNMO), human||T cells||T cell epitopes from the SARS-CoV-2 spike protein (S), nucleoprotein (N), membrane protein (M), and the open reading frame (ORF)-3a and ORF-7a proteins (O)||IFN-γ, IL-2, Granzyme B|
|PepPool: SARS-CoV-2 (S), human||T cells||T cell epitopes from the SARS-CoV-2 spike protein (S)||IFN-γ, IL-2, Granzyme B|
|PepPool: SARS-CoV-2 (NMO), human||T cells||T cell epitopes from the SARS-CoV-2 nucleoprotein (N), membrane protein (M), and the open reading frame (ORF)-3a and ORF-7a proteins (O)||IFN-γ, IL-2, Granzyme B|
|PepPool: SARS-CoV-2 (S1), scanning||T cells (scanning pool)||T cell epitopes from the S1 domain of the SARS-CoV-2 spike protein||IFN-γ, IL-2, Granzyme B|
|PepPool: SARS-CoV-2 Omicron BA.1 (S1), scanning||T cells (scanning pool)||Similar to the (S1) scanning pool, but including Omicron BA.1 mutations||IFN-γ, IL-2, Granzyme B|
Why do we have so many pools for SARS-CoV-2?
Because they can be used for different purposes and answer different questions.
PepPool: SARS-CoV-2 (SNMO), human is the go-to product for general questions on COVID-19 immunity. However, PepPool: SARS-CoV-2 (S1), scanning is preferable if you have seen that PepPool: SARS-CoV-2 (SNMO), human doesn’t have the HLA coverage you need.
A paper by the Sette lab at La Jolla (Yu et al, Cell Host & Microbe, 2022) highlighted how immune responses to vaccination and infections can be differentiated by investigating IFN-γ responses to spike and non-spike proteins, respectively. Thus, PepPool: SARS-CoV-2 (S), human can be used in combination with PepPool: SARS-CoV-2 (NMO), human to distinguish SARS-CoV-2 infection from vaccination.
Finally, PepPool: SARS-CoV-2 Omicron BA.1 (S1), scanning can be used for evaluating responses to the Omicron BA.1 mutations.
Peptide pools can be… pooled
Please note that peptide pool vials can be pooled (no pun intended) to better match the research question at hand. Maybe you only want to see TB responsiveness and aren’t interested in what specific antigen is giving the response? Maybe you are not interested in CD4 vs CD8 responses, just the total T cell response? Maybe you want to combine pools (e.g., EBV+CMV) to cover more HLA types in positive controls?
The only risk is that the DMSO concentration can become too high which leads to membrane damage and can be toxic to the cells. To address that issue, add 40 μl DMSO only the first vial, then transfer all content to the next vial, and to the next if combining three vials. Remember to rinse the vials with PBS (see image for details).
Add 40 µl DMSO to pool 1 to dissolve the peptides
Add 85 µl PBS to pool 1 to rinse the vial
Transfer the solution to pool 2 to dissolve all peptides
Transfer to pool 2 and mix
Stimuli as assay controls
Common reagents for polyclonal activation of T cells are Phytohemagglutinin (PHA) or PMA/Ionomycin. However, in our view, antigen-specific stimulation is often preferable also for control wells, as these reagents are well defined and induce a more “physiologically correct” activation of the cells. A drawback with antigen-specific stimuli is that they may not be available for all HLA types or species.
Polyclonal T cell stimulation
Selected anti-CD3 antibodies can be used as polyclonal activators of T cells. We supply monoclonal antibodies to human and monkey CD3, that elicit the release of a multitude of cytokines. The fact that a monoclonal antibody is used, produced in vitro, ensures low batch variability – an advantage over e.g., lectins.
Lectins such as PHA and Concanavalin A (ConA) are potent T cell activators. Presently, we do not offer lectins, but PHA and ConA are commercially available from many different vendors and can be purchased in varying degrees of potency.
As polyclonal stimulants activate so many cells, we recommend to lower the cell number in wells to 50,000.
Antigen-specific T cell stimulation
Antigens that are known to induce specific responses are also valuable as positive controls. A major area of use for ELISpot or FluoroSpot is the detection of IFN-γ production by antigen-specific human CD8 T cells. For this application, we have developed a positive antigen-specific control, PepPool: CEF (CD8), human.
We also supply PepPool: CEFTA (CD4), human for stimulation of CD4 T cells, and a more comprehensive version of CEF called PepPool: CEF extended (CD8), human. All three peptide pools can also be used in flow cytometry.
- PepPool: CEF (CD8), human consists of 23 viral peptides that stimulate human CD8 T cells to produce primarily IFN-γ responses in approximately 90% of all caucasians. The peptides contain MHC class I-restricted T-cell epitopes from CMV, EBV and Influenza virus, designed to stimulate T cells from donors with a variety of HLA types. It also elicits Perforin and Granzyme B responses in cells from many individuals. In addition to serving as a positive control, the CEF peptide pool is useful for standardization procedures.
- PepPool: CEF extended (CD8), human consists of 32 viral peptides that stimulate human CD8 T cells to produce primarily IFN-γ responses in approximately 90% of all caucasians. The peptides contain MHC class I-restricted T-cell epitopes from CMV, EBV and Influenza virus, designed to stimulate T cells from donors with a variety of HLA types. It also elicits Perforin and Granzyme B responses in cells from many individuals.
- PepPool: CEFTA (CD4), human consists of 35 MHC class II-restricted viral peptides from human CMV, EBV, Influenza virus, Tetanus toxin and Adenovirus 5. The CEFTA peptide pool stimulates human CD4 T cells to produce IFN-γ, IL-2, IL-5 and IL-13 and is recommended as a positive control in ELISpot and FluoroSpot assays using human PBMCs.
Depletion of CD4 and CD8 cell respectively, reveal that PepPool: CEF (CD8), human indeed predominantly stimulate CD8 T cells.
Polyclonal Monocyte/DC stimulation
Ligands for Toll Like Receptors (TLRs) can be used as positive controls to stimulate monocytes and dendritic cells. Lipopolysaccharides (LPS), purified from bacteria, act through TLR4 and are potent stimuli of e.g., IL-1β, IL-6 and TNF-α secretion. The compound R848 is a ligand for TLR7/8 and stimulates secretion of e.g., IL-1β.
Presently, we do not offer LPS, but these reagents can be acquired from multiple vendors. We do not offer R848 separately, but it is (together with IL-2) part of StimPack: Memory B cells, human, and can thus be acquired from us.
Polyclonal B cell stimulation
Memory B cells require polyclonal stimulation before secreting detectable amounts of antibody. These cells can be stimulated with StimPack: Memory B cells, human or StimPack: Memory B cells, mouse; a mixture of the synthetic compound R848 (TLR 7/8 agonist) and recombinant IL-2. Make sure to check out our step-by-step protocol for memory B cell stimulation!
|Table 2: Stimuli for assay controls|
|Stimuli||Cell type stimulated||Comment||Stimulates secretion|
|PepPool: CEF (CD8), human||CD8+ T cells (human)||Ag-specific stimulation by T cell epitopes derived from CMV, EBV and influenza.||IFN-γ, IL-2, Granzyme B, Perforin|
|PepPool: CEF extended (CD8), human||CD8+ T cells (human)||Ag-specific stimulation by T cell epitopes derived from CMV, EBV, and influenza||IFN-γ, IL-2, Granzyme B, Perforin|
|PepPool: CEFTA (CD4), human||CD4+ T cells (human)||Ag-specific stimulation by T cell epitopes derived from CMV, EBV, TT and influenza.||IFN-γ, IL-2, IL-5, IL-13|
|Anti-human CD3 mAb (CD3-2), azide free||T cells (human)||Polyclonal T cell activation||IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL-17A|
|Anti-monkey CD3 mAb (CD3-1), azide free||T cells (non-human primate)||Polyclonal T cell activation||IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL-17A|
|Phytohemagglutinin (PHA)*||T cells||Polyclonal T cell activation||IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-21, GM-CSF, Granzyme B, Perforin|
|StimPack: Memory B cells, human||B cells (human)||Polyclonal B-cell activation (R848 + recombinant hIL-2)||IgG, IgA, IgM|
|StimPack: Memory B cells, mouse||B cells (mouse)||Polyclonal B-cell activation (R848 + recombinant mIL-2)||IgG, IgA, IgM|
|Anti-human CD40 mAb (S2C6), azide free + recIL-4||B cells||Polyclonal B-cell activation||IgE|
|Lipopolysaccharide (LPS)*||Monocytes/DC||TLR4 agonist||IL-1β, IL-6, IL-8, IL-10, IL-12, IL-23, GM-CSF, TNF-α|
|R848*||Monocytes/DC||TLR7/8 agonist||IFN-α, IL-1β, IL-6, IL-23, GM-CSF, TNF-α|