*4.5. Leukocytes*

Leukocytes were rapidly isolated from venous citrated blood by lyzing erythrocytes in a solution containing 0.15 M NH4Cl, 10 mM NaHCO3, and 0.1 mM EDTA [39].

#### *4.6. Detection of Reactive Oxygen Production*

Reactive oxygen production was detected by flow cytometry, with APF, as described elsewhere [40]. Cells (200,000 cells/well) were incubated for 1 hour, with the samples (12.5, 25, 50, and 100 μg mL<sup>−</sup>1; final value). Free cells with (phosphate buffer saline) PBS, instead of the samples, were used as the negative control and were considered to be 100%. After 10 min on ice, the cells were analyzed, immediately, by a four color FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer. Forward and side scatter light was used to identify the neutrophils cell populations.

## *4.7. IL-6-, IL-10- and TNFα-Inducing Activity of CRGs and Ech on Human Blood Cells*

Blood processing was performed using the procedure described by De Groote et al. [41]. Heparinated peripheral blood was diluted 1:5 in sterile Medium 199 (Sigma, St. Louis, MO, USA) with glutamine (300 mg L−1) (Gibco, Darmstadt, Germany) and gentamicin (50 μg ml−1). Diluted blood (0.1 mL) was incubated with the investigated samples, in saline (37 ◦C, 5% CO2). The CRGs were added to obtain the final concentrations of 5, 10, and 20 μg mL−<sup>1</sup> Ech (to 1 μg mL−1) and their complexes, to obtain final concentration values corresponding to the substances, separately. After 24 h, the supernatants were collected and frozen, followed by determining the cytokine content, using specific ELISA kits, according to the manufacture's protocol ("Cytokine", Saint-Petersburg, Russia).

#### *4.8. Cell culture*

The human cancer cell line HT-29 was obtained from the American Type Culture Collection (https://www.lgcstandards-atcc.org/). HT-29 cells were incubated at 37 ◦C in a 5% CO2 humidified atmosphere, in McCoy's 5a Medium Modified, containing 10% *v*/*v* FBS (Lot RWH35894, HyClone, Logan, UT, USA), 2 mM L-glutamine, and 1% penicillin/streptomycin (Invitrogen, Paisley, UK).

## *4.9. xCELLigence System*

Experiments on the xCELLigence system were conducted by means of the Real-Time Cell Analyzer Dual Plate (RTCA-DP) instrument (ACEA Bioscience, San Diego, CA, USA). The recommendations proposed by Ke et al. [42] and Sokolova et al. [35] were applied to study the effect of the samples on human intestinal epithelial cell monolayers.

Samples were added as follows:

Single samples. Carrageenans (20 μL, *C* = 250, 500, and 1000 μg mL<sup>−</sup>1); Ech (20 μL, *C* = 10 μg mL<sup>−</sup>1). Complex samples. Carrageenan (10 μL, *C* = 500, 1000, and 2000 μg mL−1) + Ech (10 μL, *C* = 20 μg mL<sup>−</sup>1). (Concentrations are expressed as initial values.)
