The ARPE-19, adult retinal pigmented epithelial cell line, (Cellular Bank of Rio
de Janeiro, Brazil) was incubated following the methodology described by Toledo et al., 2019 (link) [25 (link)]. For cell viability, sulforhodamine B (SRB)
colorimetric assay was carried out [26 (link)].
About 10,000 cells/well were applied in 96-well plates. After 24 hours, the
cells received the peptides PnPa11 and PnPa13 at the following concentrations:
0.25; 0.5; 1.25; 2.5; 3.75; 5.0; 8.0, 12.5 and 25.0 µg/mL, and the plate was
incubated for 48hours. The medium was replaced, and the cells were fixed with
10% (v/v) trichloroacetic acid (Sigma Aldrich, USA). Subsequently, the cells
were rinsed with water and stained with 0.057% (v/v) SRB solution (Sigma
Aldrich, USA) in 1% (v/v) acetic acid (HAc) for 30 minutes at 30°C. Thereafter,
the cells were rinsed with 1% (v/v) HAc, then incubated with tris base, 110 mM,
pH 10.5 (Sigma Aldrich, USA), and shaken for 5 min. Absorbance was measured (510
nm), using a microplate reader (Bio-rad, San Diego, CA). Three wells per dose
were used in three independent experiments. The cell viability was calculated as
a percentage of the control. Besides, morphological changes in the cells were
observed (5x) using a microscope (Axio Imager M2; ZEISS, Germany).
de Janeiro, Brazil) was incubated following the methodology described by Toledo et al., 2019 (link) [25 (link)]. For cell viability, sulforhodamine B (SRB)
colorimetric assay was carried out [26 (link)].
About 10,000 cells/well were applied in 96-well plates. After 24 hours, the
cells received the peptides PnPa11 and PnPa13 at the following concentrations:
0.25; 0.5; 1.25; 2.5; 3.75; 5.0; 8.0, 12.5 and 25.0 µg/mL, and the plate was
incubated for 48hours. The medium was replaced, and the cells were fixed with
10% (v/v) trichloroacetic acid (Sigma Aldrich, USA). Subsequently, the cells
were rinsed with water and stained with 0.057% (v/v) SRB solution (Sigma
Aldrich, USA) in 1% (v/v) acetic acid (HAc) for 30 minutes at 30°C. Thereafter,
the cells were rinsed with 1% (v/v) HAc, then incubated with tris base, 110 mM,
pH 10.5 (Sigma Aldrich, USA), and shaken for 5 min. Absorbance was measured (510
nm), using a microplate reader (Bio-rad, San Diego, CA). Three wells per dose
were used in three independent experiments. The cell viability was calculated as
a percentage of the control. Besides, morphological changes in the cells were
observed (5x) using a microscope (Axio Imager M2; ZEISS, Germany).
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