*2.3. Measurement of Cell Cytotoxicity and the Production of Nitric Oxide (NO) and Prostaglandin E2 (PGE2)*

The cytotoxicity of the *Spirulina* extracts to mouse microglial cells (BV-2, ATCC, Manassas, VA, USA) was observed with the following method [42]. First, BV-2 cells were seeded in a 24-well plate at a concentration of 1 <sup>×</sup> 10<sup>5</sup> viable cells/mL with Dulbecco's modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA, USA) enriched with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), and 100 U/mL penicillin and 100 μg/mL streptomycin at 37 ◦C with 5% CO2. Then, the cells were treated with various concentrations of two samples (UE and EE) with or without 1 μg/mL

of lipopolysaccharide (LPS, L2630, Sigma, St. Louis, MO, USA), as well as with no treatment as a control, and cultured for one more day [43]. Next, the culture medium was removed and 1 mg/mL 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) solution was added. The cells were cultured again for 4 h at 37 ◦C with 5% CO2 with minimal light. The MTT solution was then removed, 200 μl of dimethyl sulfoxide (DMSO, Sigma) was added to the wells, and the wells were incubated for 30 min in the dark. After that, the absorbance was measured at a wavelength of 570 nm by a microplate ELISA reader (Thermo Fisher Scientific, Waltham, MA, USA). The cytotoxicity was estimated as the ratio of the absorbance of the untreated group (Au) to that of the sample group (As), as shown in the following Equation (2) [44]:

$$\text{Cytotoxicity (\%)} = (1 - \text{Au/As}) \times 100. \tag{2}$$

The concentrations of NO and PGE2 secreted from BV-2 cells were measured with the following method. To measure NO production from BV-2 cells, the BV-2 cells were seeded into 96-well plates at a concentration of 1.0 <sup>×</sup> 105 cells/mL, certain concentrations of each of the samples were added to the wells, and the cells were incubated for 4 h. Then 1 μg/mL of LPS was added, and the cells were cultured for 24 h at 37 ◦C in a CO2 incubator (CB150, Binder, Bohemia, NY, USA) with 5% CO2. Next, 50 μL of Griess reagent and 50 μL of cell culture supernatant were mixed, added to the cells, and allowed to react for 5 min in 96-well plates. The amount of NO in the medium was measured at 540 nm using an ELISA Reader (Thermo Fisher Scientific, Waltham, MA, USA) with NaNO2 as a standard [45]. To measure PGE2, a PGE2 EIA kit (R&D Systems, Minneapolis, MN, USA) was used by adding the supernatants from BV-2 cells treated with the same procedures described above for NO experiments. The resulting culture media were used to measure the amounts of PGE2 using an ELISA Reader (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols [46].

#### *2.4. Reverse Transcription Polymerase Chain Reaction (RT-PCR)*

To measure the expression of tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6) and Interleukin-1<sup>β</sup> (IL-1β) in murine microglial cells, 1 <sup>×</sup> 10<sup>5</sup> cells/mL of BV-2 cells were cultured with or without varying concentrations of the *Spirulina* along with 1 μg/mL of LPS, following the same procedures in the NO and PGE2 measurement experiments. After incubation, RNA was isolated from BV-2 cells using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocols. Then 1 μg of total RNA was synthesized into cDNA by a cDNA synthesis kit (RevertAid First Strand Kit, Fermentas, ON, Canada) with incubations at 25 ◦C for 5 min., 42 ◦C for 60 min., and finally 85 ◦C for 5 min. Then, 40 μl of cDNA was mixed with 0.5 μl of each target gene primer for mouse TNF- α, IL-6, IL-1β and β-actin. Forward and reverse primers were provided by the manufacture (Bioneer, Inc., Seoul, Korea) as premade primers as follows: TNF- α (N-4015, 300 bp), IL-6 (N-4013, 155 bp), IL-1β (N4009, 291 bp) and β-actin (N4021, 395 bp), respectively. The mixture was amplified with an RNA PCR kit (Takara, Shiga, Japan) at 94 ◦C for 30 s, 55 ◦C for 30 s and 72 ◦C for 1 min for 35 cycles using a PCR equipment (XP Thermal Cycler, TC-XP, BIOER Tech. Co., Hangzhou, China). Then, the PCR products were analyzed on ethidium bromide (EB, Sigma)-stained 1.2% agarose gels (BioRad co., Berkeley, CA, USA) through electrophoresis with 1-5 V/cm of the applied voltage. The amount of mRNA corresponding to each gene was quantified by Image Processing Analysis in Java (Image J, National Institute of Mental Health, Bethesda, MD, USA) by normalizing to the housekeeping gene β-actin; the values were expressed as the relative amounts (%).

#### *2.5. Measurement of the Secretioin of Pro-Inflammatory Cytokines from BV-2 Cells*

The amounts of three different Cytokines, tumor necrosis factor-α (TNF-α), Interleukin 6 (IL-6), and Interleutin-1β (IL-1β) secreted from mouse microglial cells were measured with TNF-α, IL-6 and IL-1<sup>β</sup> ELISA kits (R&D Systems) as follows [47]. First, BV-2 cells (1 <sup>×</sup> 105 cells/mL) were inoculated into a 96-well plate and cultured for one day at 37 ◦C with 5% CO2. Then, various concentrations of

the *Spirulina* extracts and 1 μM of vitamin D3 (cholecalciferol, Sigma) dissolved in 0.1% ethanol were added to 1 μg/mL of LPS for one additional day of culturing. Following the ELISA kit manuals, 50 μL of the assay diluent was added to the wells, and the standard samples were also added to the wells. Then the plate was shaken for one week and left unattended for 2 h at room temperature in the dark. Next, the plate was washed with a wash buffer, 100 μL of the substrate solution was added to the plate, and the plate was incubated for 30 min at room temperature in the dark. After blocking the reaction with a stop solution, the concentrations of the cytokines were measured at 450 nm with an ELISA reader (Thermo Fisher Scientific).
