Table 1. Multiplex assay of cell culture supernatant from ARPE-19 cells stimulated with various danger signals.
| Amyloid-ß | A2E | CEP-HSA | CEP-Lysine | CML-HSA | Vitronectin | ||
| Chemokine | IL-8 | 115.1 (62.1) | 40.4 (10.7) | 282.7 (37.6) | 40.4 (10.7) | 282.7 (37.6) | 107.1 (41.1) |
| 30.6 (21.0) | 1225.5 (24.6) | 217.8 (29.1) | 74.6 (22.0) | 328.3 (70.7) | 92.1 (14.1) | ||
| MCP-1 | 3358.0 (602.1) | 1716.0 (222.3) | 5258.8 (122.0) | 1716.0 (222.3) | 5258.8 (122.0) | 3827.9 (707.1) | |
| 2412.4 (154.8) | 7986.7 (198.6) | 4756.9 (211.2) | 2186.6 (131.9) | 4872.8 (355.4) | 3984.8 (214.6) | ||
| MIG | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 48.3 (4.9) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| MIP-1α | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 96.6 (12.5) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| MIP-1ß | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| Cytokine | G-CSF | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) |
| 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| IFN-γ | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| IL-1ß | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 76.5 (5.4) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| IL-2 | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 261.7 (9.8) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| IL-6 | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 21.4 (1.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| TNF-α | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | |
| 0.0 (0.0) | 46.5 (1.7) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | ||
| Other | VEGF-A | 673.0 (63.7) | 458.8 (44.1) | 955.3 (40.4) | 458.8 (44.1) | 955.3 (40.4) | 623.4 (106.6) |
| 485.1 (47.3) | 1169.8 (39.5) | 923.7 (32.8) | 577.6 (91.5) | 913.3 (62.9) | 635.2 (17.9) |
Cells were incubated for 24 hours with danger signals associated with AMD or the corresponding negative control. Cells were not pre stimulated with IL-1α. Cell culture supernatant was then assessed for the presence of twelve different proteins (grouped as chemokines, cytokines and other) using Multiplex assay. Concentrations of these proteins are expressed in pg/ml. Mean values (with standard deviation in brackets) are presented. The upper value represents stimulation with the negative control. The lower value represents stimulation with the molecule of interest. Each assay was performed thee times (n = 3) using three separate cell culture wells. Statistically significant rises in chemokine/cytokine/other level are highlighted in bold (all p<0.0001, one-way ANOVA).
Test molecules: Amyloid-ß (1-42) 10 µM (negative control = Amyloid-ß (42-1)), A2E (dissolved in DMSO) 25 µM (negative control = DMSO only), CEP-HSA 1 mg/ml (negative control = HSA), CEP-Lysine (dissolved in DMSO) 100 µM (negative control = DMSO only), CML-HSA 1 mg/ml (negative control = HSA), Vitronectin 100 nM (negative control = Nil).
AMD = Age related macular degeneration, A2E = N-retinylidene-N-retinylethanolamine, CEP-HSA = Carboxyethylpyrrole modified human serum albumin, CEP-Lysine = Carboxyethylpyrrole modified lysine, CML-HSA = Nε-(Carboxymethyl)lysine modified human serum albumin, DMSO = Dimethyl sulphoxide, HSA = Human serum albumin.