Overview cell stimuli in ELISPOT assays

Overview general cell stimuli commonly used in ELISPOT and FluoroSpot assays

The below mentioned stimuli for T cells, monocytes and dendritic cells can be used in T cell ELISPOT assays and FluoroSpot assays as positive controls to demonstrate that the cells are functional and the assay works fine.

The mentioned stimuli for B cells can be used in B cell ELISPOT assays to activate memory B cells.

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, G-CSF	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, IL-21, G-CSF, GM-CSF	2x10E5 PBMC/well	0.5 LF/ml
Phytohaemagglutinin (PHA)	polyclonal	T cells	IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-21, G-CSF, GM-CSF, Granzyme B, Perforin, TNF-α	2x10E3 to 1x10E5 cells/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, 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-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)	polyclonal	Human/non-human primate monocytes/ dendritic cells	IL-6, IL-10, GM-CSF	1x10E3 to 5x10E4 PBMC/well	LPS: 100 ng/ml
Lipopolysaccharide (LPS) + IFN-γ	polyclonal	Human/non-human primate monocytes/ dendritic cells	IL-1β, IL-6, IL-10, IL-12, GM-CSF	1x10E3 to 5x10E4 PBMC/well	LPS: 100 ng/ml + IFN-γ: 1-100 units/ml
R848 + IL-2	polyclonal	B cells	IgG	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

NOTE: 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).