*5.5. Cell Assays*

#### 5.5.1. Murine Peritoneal Macrophages Obtainment

Male young, between 8 and 12 weeks of age and weighing between 20 and 22 g, BALB/c mice adults were used. Mice were obtained from the Central Animal Facility of the Butantan Institute and housed in the Laboratory of Immunochemistry bioterium, Butantan Institute. The mice were kept in boxes lined with shavings, containing 3 animals per box, under natural light, full-time ventilation and exhaustion, filtered water and commercial feed ad libitum. After a period of 2 to 3 days of acclimatization of the animals, they were euthanized in a CO2 chamber. All experimental procedures involving animals were in accordance with the ethical principles in animal research adopted by the Brazilian Society of Animal Science and the National Brazilian Legislation no.11.794/08. Animal care and experimental procedures were approved by the Institutional Committee for the Care and

Use of Laboratory Animals from Butantan Institute (CEUAIB protocol number 5396310517, approved on 21 June 2017).

Peritoneal exudate cells (BALB/c naïve mice, *n* = 6) were collected by two washes with 5 mL of RPMI medium. The cells obtained were washed twice with RMPI medium, at 400 g, for 10 min, at 18 ◦C, and resuspended in R10 medium (RPMI + 10% fetal cow serum). After counting in a Neubauer chamber, in the presence of Trypan blue, the cell concentration was adjusted to 2 × <sup>10</sup><sup>6</sup> cells/mL. Cells were distributed into 96-well culture plates (2 × 105 cells/100 <sup>μ</sup>L/well) and incubated for 2 h at 37 ◦C and 5% CO2. After incubation, non-adherent cells were discarded, and adherent cells (macrophages) were resuspended in R10 medium containing the respective tested stimuli, at different concentrations, in triplicates, in a final volume of 200 μL/well. Cells resuspended in R10 medium were used as a negative control, and cells stimulated with LPS (5 μg/mL) as a positive control. Cells were then incubated for 24 h at 37 ◦C and 5% CO2. After this period, the culture supernatants were collected and stored at −80 ◦C, for subsequent dosage of cytokines, and cell viability was determined by MTT assay.

5.5.2. Effect of Hypotensins on the Cell Viability and Production of Inflammatory Mediators by Murine Peritoneal Macrophages Stimulated In Vitro with Hypotensins

After a 24-h period of incubation of cells with hypotensins (10 μg/mL, 50 μg/mL and 100 μg/mL), MTT assays were performed, consisting of the addition of 100 μL of R10 containing 0.5 mg/mL of tetrazolium salt 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in all wells, followed by a new incubation, under the same conditions, for 4 h. Then, the supernatants were discarded, and 100 μL/well of DMSO were added to dissolve the formed crystals. Absorbance was determined in a spectrophotometer at 540 nm. Cell viability was calculated based on the absorbances of the samples and the negative control (cells cultivated only with R10).

The concentration of pro- and anti-inflammatory cytokines (IL-6, IL-10, IL-12p70, IFN-α, MCP-1 and TNF) in the samples incubated with both hypotensins (10 μg/mL, 50 μg/mL and 100 μg/mL) was determined by the CBA method (BD Cytometric Bead Array Mouse Inflammation Kit), according to the manufacturer's instructions. The samples were acquired in a BD FACSCanto II flow cytometer, and the data were analyzed using BD FCAP Array version 3.0 software.
