Melanoma cells were seeded in 96-well plates (12.0 × 103 cells/well for each cell line). The day after, cells were treated with HPF at the concentration of 3 µM, followed by an incubation of 48 h. At the end of the treatment, cells were fixed by adding 25 µL/well of 50% (w/v) trichloroacetic acid into the culture medium. Plates were then incubated at 4 °C for 1 h, washed 4 times with deionized water (ddH2O), and allowed to dry at room temperature (RT). To perform staining, 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy) was added. After incubating for 1 h at RT, the plates were rinsed with 1% acetic acid and allowed to air dry. SRB stain was then solubilized using a 10 mM Tris-base solution at pH 10.5. Subsequently, the absorbance of the samples was measured at 540 nm using a TECAN NanoQuant Infinite M200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland). Five to six replicates were performed for each condition or data point.
Sulforhodamine b srb sodium salt solution
Manufactured by Merck Group
Sourced in Italy
Sulforhodamine B (SRB) sodium salt solution is a laboratory reagent used for colorimetric assays. It is a red-colored dye that binds to basic amino acid residues of proteins, allowing for the quantification of cellular protein content. The solution is typically used in cell proliferation and cytotoxicity assays.
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4 protocols using sulforhodamine b srb sodium salt solution
1
Sulforhodamine B Assay for Melanoma Cells
2
Sulforhodamine B Cytotoxicity Assay
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Melanoma MeWo and A375 cells or NHEM were seeded in 96-well plates (5.0 × 103 cells/well for MeWo cells and 2.9 × 103 cells/well for A375 or NHEM). After 24-h culture, the cells were treated with different concentrations of compounds 1 –7 . After 72-h treatment, the cells were fixed by adding 25 µL/well of 50% (w/v) TCA directly into the culture medium. The plates were incubated at 4 °C for 1 h, washed four times with ddH2O, and dried at RT. Staining was performed by adding 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy). After 1-h incubation at RT, the plates were rinsed with 1% HAc and air-dried. SRB was solubilized in a 10 mM Tris-base solution pH 10.5 and Abs492 measured in the plate reader TECAN NanoQuant Infinite M200 Pro (Tecan Group Ltd., Männedorf, Switzerland). Six replicates for each condition/data point were performed.
3
Cytotoxicity Evaluation of HPF
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A375, FO1, SK-Mel-28 and NHEM cells were seeded in 96-well plates (A375: 3.0 × 103 cells/well; FO1, SK-Mel-28, MeWo and NHEM: 6.0 × 103 cells/well). After 24 h, cells were treated with different concentrations of HPF and incubated for 24, 48 and 72 h. At the end of each treatment, cells were fixed by adding 25 µL/well of 50% (w/v) trichloroacetic acid directly into the culture medium. Plates were incubated at 4 °C for 1 h, washed 4 times with ddH2O and dried at room temperature (RT). Staining was performed by adding 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy). After 1 h incubation at RT, plates were rinsed with 1% acetic acid and air-dried. SRB was solubilized in 10 mM Tris base solution, pH 10.5 and Abs 540 nm, measured in the plate reader TECAN NanoQuant Infinite M200 Pro (Tecan Group Ltd., Männedorf, Switzerland). Six replicates for each condition/data point were performed.
4
Sulforhodamine B Assay for Melanoma Cell Viability
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Melanoma cells were plated in 96-well plates (SK-Mel-28: 4.0 × 103 cells/well; A375: 2.9 × 103 cells/well; FO-1: 3.5 × 103 cells/well; MeWo: 6 × 103 cells/well; NHEM: 6 × 103 cells/well). The following day, the cells underwent treatment with antiretroviral drugs or the antineoplastic alkylating agent DTIC (MERK, Milan, Italy) with an incubation period of 72 h. Upon completion of the treatment, cell fixation was achieved by adding 25 µL/well of 50% (w/v) trichloroacetic acid to the culture medium. Subsequently, the plates were incubated at 4 °C for 1 h, washed four times with deionized water (ddH2O), and left to air-dry at room temperature (RT).
For staining, 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy) was applied. After a 1-h incubation at RT, the plates were rinsed with 1% acetic acid and allowed to air-dry. The SRB stain was then solubilized using a 10 mM Tris-base solution at pH 10.5. Subsequently, the absorbance of the samples was measured at 540 nm using a TECAN NanoQuant Infinite M200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland). Each condition or data point was subjected to four to eight replicates.
For staining, 50 µL/well of 0.04% (w/v) sulforhodamine B (SRB) sodium salt solution (Sigma-Aldrich, Milan, Italy) was applied. After a 1-h incubation at RT, the plates were rinsed with 1% acetic acid and allowed to air-dry. The SRB stain was then solubilized using a 10 mM Tris-base solution at pH 10.5. Subsequently, the absorbance of the samples was measured at 540 nm using a TECAN NanoQuant Infinite M200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland). Each condition or data point was subjected to four to eight replicates.
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