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Guidelines for stimuli, cell concentrations and cell incubation times in T cell ELISPOT and FluoroSpot assays

Guidelines for stimuli, cell concentrations and cell incubation times

in T cell ELISPOT and FluoroSpot assays

 

The stimuli listed below for T cells, monocytes and dendritic cells can be used in T cell ELISPOT and FluoroSpot assays as positive controls to demonstrate that the cells are capable to produce cytokines, and that the ELISPOT or FluoroSpot assay was performed properly.

Notes:

  • Optimal concentrations and incubation times should be determined experimentally, especially when working with heterogeneous cell populations. Please take into account that different cell types can produce the same effector molecules but require different conditions for stimulation. For example, the optimal conditions for the detection of IFN-γ secreting CD8+ T cells in PBMC suspensions differ significantly from those for the detection of IFN-γ secreting CD4+ T cells (Schmittel et al. 2001). Moreover, the production of cytokines, such as TNF-α, IL-6 and IL-10, is not restricted to T cells and many spot forming cells can also be attributed to activated monocytes/macrophages. Adherence of this last cell type to the surface of an ELISPOT or FluoroSpot well may already be sufficient to trigger TNF-α and IL-6 release. 
  • For all assay controls, the same procedure and incubation times should be followed as accounts for the antigen-specific stimulation of the experimental antigen of interest. The only difference between the positive controls and the antigen-specific stimulation/negative control may be a lower final cell concentration per well on the ELISPOT plate to avoid confluent or poorly defined spots.
  • Detailed information on cell preparation and the ins and outs on preincubation see: Cell sample preparation.

 

ELISPOT assay

Cell type: Human and Old World Monkey PBMCs
Analyte Recommended stimuli (see below) Incubation time on ELISPOT plate without preincubation Incubation time on ELISPOT plate after preincubation
IFN-γ 1-4,7,8 24-48 h 24 h
IL-1β 1,6
24-48 h (advice: resting cryopreserved cells) 24 h
IL-2 1-4,7,8 24-48 h 24 h
IL-4 1-4,8 48 h 24 h
IL-5 1-4,8 48 h 24 h
IL-6 2,5,6 24-48 h (advice: resting cryopreserved cells) 24 h
IL-10 1-5,6,8 48 h 24 h
IL-12p70 6 24-48 h preincubation not recommended
IL-12/23p40 6 24-48 h 24 h
IL-13 1-4,8 48 h 24 h
IL-17A 1-4,8 24-48 h 24 h
IL-17F 1 24-48 h 24 h
IL-21 1-3,8 48 h 24 h
G-CSF 2,3,7,8 24-48 h 24 h
GM-CSF 1-3,5,6,8 24-48 h 24 h
Granzyme B 1-4 48-72 h 24 h
Perforin 1,2 preincubation recommended 24 h
TNF-α 1-3 24-48 h 24 h

Notes:

  • Preincubation time: 24-48 h at a cell concentration of 4x106 cells/ml.
  • Cell concentration antigen stimulation in ELISPOT well: 2x105 cells/well.
  • Cell concentration polyclonal stimulation in ELISPOT well: 2x103 to 1x105 cells/well.
  • Resting time of cryopreserved cells: 18-24 h.
 
Cell type: New World Monkey PBMCs
Analyte Recommended stimuli (see below) Incubation time on ELISPOT plate without preincubation Incubation time on ELISPOT plate after preincubation
IFN-γ 1-3 24-48 h  
IL-12/23p40 6 24-48 h  
IL-13 1,2 48 h  
IL-17A 2 24-48 h  
TNF-α 2 24-48 h  

Notes:

  • Preincubation time: 24-48 h at a cell concentration of 4x106 cells/ml.
  • Cell concentration antigen stimulation in ELISPOT well: 2x105 cells/well.
  • Cell concentration polyclonal stimulation in ELISPOT well: 2x103 to 1x105 cells/well.
 
 Cell type: Rodent spleen cells
Analyte Recommended stimuli (see below) Incubation time on ELISPOT plate without preincubation Incubation time on ELISPOT plate after preincubation
IFN-γ 1-3 24-48 h 24 h
IL-2 1-3 24-48 h 24 h
IL-4 1-3 48 h 24 h
IL-5 1-3 48 h 24 h
IL-10 1-3 48 h 24 h
TNF-α 1-3 24-48 h 24 h

Notes:

  • Preincubation time: 24-48 h at a cell concentration of 4x106 cells/ml.
  • Cell concentration antigen stimulation in ELISPOT well: 2x105 cells/well.
  • Cell concentration polyclonal stimulation in ELISPOT well: 2x102 to 1x105 cells/well.

 

FluoroSpot assay

Cell type: Human and Old World Monkey PBMCs
Analyte Recommended stimuli (see below) Incubation time on FluoroSpot plate without preincubation Incubation time on FluoroSpot plate after preincubation
IFN-γ / IL-1β 1 24-48 h (advice: resting cryopreserved cells) 24 h
IFN-γ / IL-2 1-4,7,8 24-48 h 24 h
IFN-γ / IL-4 1-4,8 48 h 24 h
IFN-γ / IL-5 1-4,8 48 h 24 h
IFN-γ / IL-10 1-4,8 48 h 24 h
IFN-γ / IL-13 1-4,8 48 h 24 h
IFN-γ / IL-17A 1-4,8 24-48 h 24 h
IFN-γ / Granzyme B 1-4 48-72 h 24 h
IFN-γ / TNF-α 1-3 24-48 h 24 h

Notes:

  • Preincubation time: 24-48 h at a cell concentration of 4x106 cells/ml.
  • Cell concentration antigen stimulation in FluoroSpot well: 2x105 cells/well.
  • Cell concentration polyclonal stimulation in FluoroSpot well: 2x103 to 1x105 cells/well.
  • Resting time of cryopreserved cells: 18-24 h.

 

General cell stimuli commonly used in T cell ELISPOT and FluoroSpot assays

Polyclonal stimuli:

  1. Phorbol 12-myristate 13-acetate (PMA) & ionomycin (50 ng/ml & 1 µg/ml)
  2. Phytohaemagglutinin (PHA) (10-30 ng/ml)
  3. Concanavalin A (human and non-human primate: 6-10 µg/ml) (rodent: 4 µg/ml)
  4. anti-CD3 & anti-CD28 monoclonal antibodies (0.05 µg/ml & 0.05 µg/ml; cat. no. CT372)
  5. Lipopolysaccharide (LPS) (100 ng/ml)
  6. Lipopolysaccharide (LPS) & IFN-γ (100 ng/ml & 10-100 units/ml)

Antigen-specific stimuli (for human PBMC only):

  1. ICE peptide pool (a pool of synthetic peptides of Influenza A, Cytomegalovirus and Epstein-Barr virus; 1 µg/ml for each peptide; cat. no. CT387)
  2. Tetanus toxoid (0.5 LF/ml)

 

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