Srb solution

Additionally, 96-well plates were seeded with MCF-7, HeLa, and HePG2 cells, at approximately 105/well. Following 72 h exposure to honey and honey with AgNPs, the medium was changed to 150 L of 10% trichloroacetic acid (TCA) from Merck for 1 h at 4 °C; then, the cultures were washed five times with distilled water. Afterward, a 70 μL SRB solution (0.4% w/v) (Sigma Aldrich, St. Louis, MO, USA) was added for 10 min at room temperature in a dark location. The cells were washed three times with 1% acetic acid (Merck) and left overnight to air dry. The protein-bound SRB stain was dissolved by adding 150 μL of 10 mM Tris Base (Merck), and the O.D. was measured at 540 nm using a FluoStar Omega microplate reader (BMG Labtec, Ortenberg, Germany) [51 (link)].

Cells were seeded in 96-well tissue plates at a density of 4 × 10⁴ cells in 200 μl medium per well. Compounds were added at the indicated concentrations or wells were left untreated as controls, and the plates were incubated for 24 h. Viable cells were detected using the SRB colorimetric method. Briefly, 150 μl 50% trichloroacetic acid (Sigma) was added to each well and the plates were incubated at 4 °C for 1 h. The plates were then washed five times with ddH₂O, and then air-dried. SRB solution (50 μl/well; Sigma) was added, and the plates were incubated for 30 min at room temperature. The plates were washed five times with 1% acetic acid, air dried, and treated with 10 mM Tris base (pH 10). Absorbance was measured using a microplate reader at 565 nm.

Sulforhodamine B (SRB) colorimetric assay was performed to evaluate clonogenicity after treatment with our ADCs according to a published protocol^{53 (link)}. HCC1954 and HCC1954-TDR cells were plated into 24-well plates (1000 cells per well) and incubated overnight. The cells were treated with each ADC for 5 days. Subsequently, cells were fixed with 5% trichloroacetic acid and then stained with 0.03% of SRB solution (Sigma) at room temperature for 30 min. The stained cells were imaged using a GelCount system (Oxford Optronix) and then dissolved in Tris buffer (10 mM). Optical density was determined fluorometrically using a VICTOR X3 plate reader (Ex: 488 nm, Em: 585 nm).

The cytotoxic activity of JA against MCF-7^ADR cells was assessed by sulphrhodamine B (SRB) assay. Cells were seeded in 96-well plates, approximately 5 × 10³/well, and treated with JA at a concentration of (100, 10, 1.0, 0.1, and 0.01 µM) for 72 h. After that, the media were discarded, and 150 µL of 10% trichloroacetic acid (TCA)/ well were added (Merck) for 1 h in a fridge. Then, the cells were washed with tap water three times. Afterward, 70 µL of SRB solution (0.4% w/v) (Sigma‐Aldrich) was added for 10 min in a dark place at room temperature. The cells were first rinsed with 1% acetic acid three times and then air-dried overnight. Next, 150 µL of 10 mM Tris Base was added to dissolve the protein-bound stain. Finally, the absorbance of the solution was measured at 540 nm using a FluoStar Omega microplate reader (BMG Labtec, Ortenberg, Germany). The dose-response curve of JA was analyzed using the Emax model, as described in a previous publication [29 (link)].

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).

The cell viability was determined following the method reported by Skehan et al.²⁶ . MCF-7, MCF7^ADR, H9C2 and BNL cells were seeded in 96‐well plates, approximately 10⁵/well. After treatment of DOX and/or P. niruri fractions and subfractions with concentrations of 0.01, 0.1, 1, 10 and 100 ug/ml for 72 h. We replaced the media with 150 μL of 10% TCA (trichloroacetic acid) (Merck) for 1 h at 4 °C, followed by 5 timed washing with distilled water. After that, we added 70 μL of 0.4% w/v SRB solution (Sigma‐Aldrich) at room temperature for ten minutes in the dark. Cells were washed with 1% acetic acid (Merck) 3 times and allowed to a 24 h air-dry. Then, we added 150 μL of Tris Base (10 mM) (Merck) and the O.D. was determined at 540 nm by a microplate reader (FluoStar Omega, BMG Labtec, Ortenberg, Germany).

The viability of cells isolated from metastatic malignant melanoma tissues treated with 1,25(OH)₂D₃, vemurafenib or both was assessed using sulforhodamine B (SRB) as described previously [17 (link),30 (link),79 (link)]. The cells were seeded in 96-well plates at a density of 2000 of cells per well and left overnight. Subsequently, the cells were treated simultaneously with serial dilutions of vemurafenib (3, 15–200 nM) and 1,25(OH)₂D₃ at a 100 nM or 1 µM concentration for 72 h. The cells were fixed with 10% trichloroacetic acid for 1 h at 4 °C, washed with distilled water and stained for 15 min with 0.4% SRB solution (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) in 1 % acetic acid. SRB dye was solubilised using a solution of 10 mM buffered Tris Base (pH 10.5), and absorbance was measured at 570 nm using an Epoch spectrophotometer (BioTek, Winooski, USA). The relative IC50 value was calculated as described previously [30 (link)] as the mid-point between no inhibition and the maximum observed decrease in proliferation (n = 6).