Overview cell stimuli

The concentrations listed in the tabel below should be considered as general guidelines. It is recommended to evaluate a range of concentrations to determine the optimal stimulation conditions for your ELISPOT assay. Up to 3×10⁵ cells can be added per well in a 96-well ELISPOT plate. However, the number of cells should not be so high that spot counting becomes difficult. For reliable analysis, a spot count of approximately 50 to 100 spots per well is generally recommended.

Stimulus	Type of stimulation	Target cell type	Typical cytokines detected	Recommended cell number	Recommended concentration
ICE peptide pool (Influenza A, CMV and EBV epitopes) cat.no. CT387	antigen specific	human CD8⁺ T cells	IFN-γ, IL-2, Granzyme B, G-CSF	2x10E5 PBMC/well	1 µg of each peptide/ml
Tetanus toxoid	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
Phytoheagglutinin (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-⍺	2x10E3 to 5x10E4 PBMC/well	10-30 µg/ml
Phorbol 12-myristate 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-⍺	2x10E3 to 1x10E5 PBMC/well	PMA: 50 ng/ml + ionomycin: 1 ug/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-⍺	2x10E3 to 1x10E5 PBMC/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-y	polyclonal	human/non-human primate monocytes/dendritic cells	IL-1β, IL-6, IL-10, IL-12p70, IL-12/IL-23 p40, GM-CSF, TNF-⍺	1x10E3 to 5x10E4 PBMC/well	LPS: 100 ng/ml + IFN-γ: 1-100 units/ml
R848 + IL-2 (available on request)	polyclonal	B cells	IgG, IgM	2x10E3 to 1x10E5 PBMC/well	items are part of our B cell ELISPOT kit