Overview cell stimuli in ELISPOT assays

 

Overview general cell stimuli commonly used in ELISPOT and FluoroSpot assays


Stimuli
Type of stimulation Activated cell type Stimulates secretion of Recommended cell concentration* Recommended stimulus concentration*
ICE peptide pool (Influenza A, CMV and EBV epitopes)
(cat. no. CT387)
antigen specific Human CD8+
T cells
(Caucasian)
IFN-γ, IL-2, Granzyme B,
Perforin
2x10E5 PBMC/well 1 µg of each peptide/ml
Tetanus toxoid (TT) antigen specific Human CD4+
T cells
IFN-γ, IL-2, IL-4, IL-5, IL-10,
IL-13, IL-17A
2x10E5 PBMC/well 0.5 LF/ml
Phytohaemagglutinin (PHA) polyclonal T cells IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A,
IL-21, G-CSF, Granzyme B, Perforin, TNF-α
2x10E3 to 1x10E5 PBMC/well 10-30 µg/ml
Phorbol 12-myrstate 13-acetate (PMA)
+
Ionomycin
polyclonal T cells IFN-γ, IL-1β,
IL-2, IL-4, IL-5,
IL-6, IL-10,
IL-13, IL-17A,
IL-17F, IL-21,
G-CSF, GM-CSF, Granzyme B,
Perforin, TNF-α
2x10E3 to 1x10E5 cells/well
PMA: 50 ng/ml
+
Ionomycin:
1 µg/ml
Concanavalin A
(ConA)
polyclonal T cells IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A,
IL-21, G-CSF,
GM-CSF, Granzyme B,
TNF-α
2x10E3 to 1x10E5 cells/well human/non-human primate PBMC:
6-10 µg/ml
rodent splenocytes:
4 µg/ml
anti-CD3/CD28 monoclonal antibodies (cat. no. CT372) polyclonal Human T cells IFN-γ, IL-2, IL-4, IL-5, IL-10,
IL-13, IL-17A,
Granzyme B
2x10E3 to 5x10E4 PBMC/well 0.05 µg/ml
Lipopolysaccharide (LPS) + IFN-γ polyclonal Human monocytes/
dendritic cells
IL-1β, IL-6, IL-10, IL-12, IL-23,
GM-CSF
1x10E3 to 5x10E4 PBMC/well LPS: 100 ng/ml
+
IFN-γ: 1-100 units/ml
R848 + IL-2 polyclonal B cells IgA, IgG, IgM 2x10E3 to 1x10E5 cells/well item is part of our B cell ELISPOT kits
anti-CD40 monoclonal antibody + IL-4 polyclonal B cells IgE 2x10E3 to 1x10E5 cells/well item is part of our B cell ELISPOT kits

*) The above mentioned concentrations are guidelines. It is recommended to analyze a series of dilutions to determine the optimal concentration first. A maximum of 3x10E5 cells can be put into a well of a 96-well plate. However, it is important that the number of cells per well is not too numerous in order to facilitate counting of spots (50 to 100 spots/well).

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