Table 3.
Methods addressing several MIE’s and KE’s shown in Fig. 1
| Event | In vitro method | Read-outs |
|---|---|---|
| MIE 1, 2, 3 & KE1 | M cells |
Allergen quantification (SDS-PAGE, Western blot and microscopy) Integrity of ZO-1 (microscopy). |
| T84 | Monolayer integrity (TEER) Cytokine production (ELISA) | |
| HCT-8 | Monolayer integrity (TEER) Cytokine production (ELISA) | |
| Caco-2 |
Monolayer integrity (TEER and Lucifer Yellow) Allergen quantification (ELISA, SDS-Page, Western blot and LC–MS,) Integrity of A20 (Western blot and RT-qPCR blot and microscopy) Allergen transport (RBL activation test) Gene expression (RT-qPCR) |
|
| HT-29 |
Monolayer integrity (TEER). Allergen quantification (ELISA) Integrity of A20 (Western blot and RT-qPCR |
|
| KE 2 & 3 | Mouse BM-DCs |
Allergen uptake (flow cytometry) Migration assay (flow cytometry) Cytokine production (ELISA) DCs maturation (flow cytometry) |
| THP-1-derived DCs |
Allergen uptake (flow cytometry) Gene expression (RT-qPCR) Cytokine production (ELISA) |
|
| Human Mo-DCs | Expression of DC markers (flow cytometry) | |
| KE 4 | Human T cell clones |
T cell proliferation ([3H]-thymidine) Cytokine production (ELISA) T cell activation (flow cytometry) |
| Human peripheral blood mononuclear cells (PBMCs) |
T cell proliferation (CFSE or [3H]-thymidine) Cytokine production (flow cytometry) T cell activation (flow cytometry) Gene expression (RT-qPCR) Expression of T cell markers (flow cytometry) |
|
| Mouse MLN-isolated T cells | Cytokine production (ELISA) | |
| Mouse LP-isolated mononuclear cells |
T cell proliferation (CFSE) Cytokine production (flow cytometry) |
|
| KE 2, 3 & 4 | Co-culture: BM-DCs/primed T cells |
Allergen uptake (flow cytometry) T cell cytokine production (ELISA) DCs maturation (flow cytometry) |