Table 1.

Percent change of NF-κB in HEK cell-lines with specific Toll-like receptors (TLRs) when stimulated with alginate in the different steps of purification. Contaminations can activate hTLR2, 4, 5 and 9. Values are presented as percentages. Each cell-line was stimulated with an appropriate positive control with different values of NF-κB activation: (a) TLR2 (FSL-1, 1 μg/mL, 1.272 ± 0.057); (b) TLR3 (Poly(I:C) HMW, 5 μg/mL, 0.217 ± 0.015); (c) TLR4 (LPS-EK Ultrapure, 100 ng/mL, 0.270 ± 0.009); (d) TLR5 (RecFLA-ST, 100 ng/mL, 2.626 ± 0.023); (e) TLR7 (Imiquimod (R837), 50 μg/mL, 0.154 ± 0.056); (f) TLR8 (ssRNA 40, 50 μg/mL, 0.065 ± 0.009); (g) TLR9 (ODN 2006, 100 μg/mL, 1.088 ± 0.051); (h) NOD1 (Tri-DAP, 10 μg/mL, 0.458 ± 0.068); (i) NOD2 (L18-MDP, 100 ng/mL, 0.504 ± 0.069). DMEM 1640 growth media was used in all the cell lines as a negative control. The threshold for biologically relevant was higher than 0.01 (arbitrary units). This level is the minimum value that purified alginate in solution can reach. Values are presented as percent change; p < 0.05 (*) and p < 0.01 (**).

HEK cell-line	Purification steps							Positive control
	0	1	2	3	4	5	6
TLR2	−20.2	143.7 *	149.6 *	108.4 *	115.1 *	155.1 *	−30.8	1461.7
TLR3	0.5	−12.2	2.9	2.6	4.4	6.2	4.9	418.8
TLR4	108.1 **	70.5 **	43.4 **	38.0 **	43.0 **	65.2 **	67.7 **	324.1
TLR5	20.1 **	−3.8	28.7 **	1.8	−8.3	12.0	−4.5	1878.9
TLR7	4.86	11.15	−5.42	−4.83	0.58	1.90	−0.85	104.10
TLR8	−31.64	−1.83	6.00	−6.74	−1.22	4.74	−28.35	11.11
TLR9	6.35	2.40	−1.52	−2.46	−2.13	5.83	103.5 **	1033.33
NOD1	7.18	−5.42	−1.00	−0.72	1.28	2.53	4.13	818.94
NOD2	−4.44	−12.00	−13.47	−11.28	−14.45	−10.87	−7.46	451.05