## 2 Materials and Methods
### Immunomodulatory Properties  
A total of 10 healthy subjects (6 males and 4 females, 26-40 years old, mean age 31, non-smokers) were recruited for this study. None of the subjects had received any therapy in the 2 months prior to taking a blood sample. Peripheral venous blood samples were collected from the cubital vein in BD Vacutainer(r) K2 EDTA tubes (Becton, Dickinson and Company, Oakville, ON, Canada) in a clinical laboratory. The basic haematological parameters of all volunteers were within the reference ranges.  
Blood samples were centrifuged at 1500\(\times\)\(g\) for 15 min at room temperature, and the serum was discarded. Red blood cells were lysed with 0.84% NH\({}_{4}\)Cl buffer, and the samples were washed twice with a sterile Dulbecco's Phosphate-Buffered Saline (D-PBS, Gibco(r), Life Technologies(tm), Paisley, Scotland, UK). The isolated leukocytes were centrifuged at 1500\(\times\)\(g\) for 10 min, and the cells were resuspended in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, and stabilized with antibiotic, antimycotic solution (all from Sigma-Aldrich Chemie GmbH, Steinheim, Germany). This medium was denoted as complete DMEM. Cells were plated (1 \(\times\) 10\({}^{6}\) cells/mL) in 12-well plates (TPP, Trasadingen, Switzerland) and treated for 48 h with 4 mL (5 mg/mL) of the cyanobacterial extract to reach a final concentration of 20 mg/mL in complete DMEM (1 mL/well). Our pilot studies for cytotoxicity with the cell lines A549 (epithelial lung carcinoma cells), HT-29 (epithelial colon adenocarcinoma cells), HeLa (epithelial cervix adenocarcinoma cells), U-937 (human histiolytic lymphoma cells), and NIH/3T3 (embryonic mouse fibroblasts) showed that concentrations of the extract up to 100 mg/mL do not have any cytotoxic effects. Untreated cells cultured for 48 h in complete DMEM served as a negative control. Another control included cells treated with an equivalent amount of DMSO (4 mL of DMSO/water, 1:1) as that contained in the extracts. Cells incubated for 48 h with 1 mg/mL leucoagglutinin PHA-L (Sigma-Aldrich, Saint Louis, MO, USA) were used as a positive control. The leukocytes were cultured in complete DMEM at 37 \({}^{\circ}\)C, 5% CO\({}_{2}\), 95% atmospheric air, in a humidified incubator.  
After 48 h, the treated and control cells were collected by centrifugation at 1500\(\times\)\(g\) for 10 min, and the cell pellets were resuspended in FACS buffer (D-PBS, supplemented with 5% fetal bovine serum and 0.05% NaN\({}_{3}\)). The cells were stained for 20 min at 4 \({}^{\circ}\)C with the following fluorochrome-conjugated, anti-human antibodies grouped in 3 panels: (I) CD3 PE-Dazzle(tm) 594, CD4 FITC, CD8 PE, CD25 PE-Cy5, and CD152 PE-Cy7; (II) HLA-DR-DP FITC, CD19 PE, CD80 PE-Dazzle(tm) 594, CD11b PE-Cy5, and CD138 PE-Cy7; and (III) CD3 PE-Dazzle(tm) 594, CD56 FITC, and CD16 PE (all from BioLegend(r), San Diego, CA, USA). Finally, the cells were washed twice and resuspended in 300 \(\mu\)L FACS buffer. Immunophenotyping was performed by flow cytometry (FACS) using a Cytomics FC500 instrument (Beckman Coulter Inc., Life Sciences, Indianapolis, IN, USA). The results are presented as mean \(\pm\) SD (\(n\) = 10).  
Production of IL-2, IL-6, and TNF-\(\alpha\) in the supernatants was assayed by LEGEND MAX(tm) Human IL-2, IL-6, and TNF-\(\alpha\) ELISA kits (BioLegend Inc., San Diego, CA, USA), according to the manufacturer's instructions. The sensitivity of the assay for IL-2, IL-6, and TNF-\(\alpha\) was 4 pg/mL, 1.6 pg/mL, and 3.5 pg/mL, respectively.  
The study was conducted in adherence to the Declaration of Helsinki ethical guidelines and was approved by the Local Ethics Committee at the Paisii Hilendarski University of Plovdiv, Bulgaria. Written informed consent was obtained from all participants prior to the initiation of the study.