Liposomes were prepared by the thin-film hydration method, as reported3 (link). In brief, the lipid formulation [DSPC (Avanti Polar Lipids, Alabaster, AL, USA), DLPC (Avanti Polar Lipids, Alabaster, AL, USA), DSPG (Genzyme, Cambridge, MA, USA), and cholesterol (Sigma, St. Louis, MO, USA) at molar ratio 3:3:2:3], along with the indicated amount of the sonosensitizer PPIX, was dissolved in a solution of chloroform:methanol 9:1. The solvent was evaporated under reduced pressure, and the lipid was redissolved in t-butanol, followed by freeze-drying. The lipid cake was hydrated with PBS, TTX solution (0.375 mg/mL PBS; Abcam, Cambridge, MA, USA), or sulforhodamine B solution (10 mg/mL PBS; Aldrich, St. Louis, MO, USA). After 10 freeze–thaw cycles, the solution was dialyzed against PBS for 48 h in a dialysis tube with a molecular mass cut-off of 1000 kDa. The dialysis media were changed with fresh PBS at least twice a day. Lipo-DMED was made following the same procedure, but with PPIX not included in the formulation and with hydration in 1 mg/mL of DMED in PBS solution.
Sulforhodamine b solution
Manufactured by Merck Group
Sourced in United States
Sulforhodamine B solution is a fluorescent dye used as a labeling reagent in various research applications. It is a bright pink, water-soluble dye that can be used to stain proteins and cells. The solution is typically used in colorimetric assays, cell viability studies, and other biochemical procedures that require a fluorescent label.
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7 protocols using sulforhodamine b solution
1
Liposome Encapsulation of Sonosensitizer PPIX
2
Liposome Encapsulation of Sonosensitizer PPIX
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Liposomes were prepared by the thin-film hydration method, as reported3 (link). In brief, the lipid formulation [DSPC (Avanti Polar Lipids, Alabaster, AL, USA), DLPC (Avanti Polar Lipids, Alabaster, AL, USA), DSPG (Genzyme, Cambridge, MA, USA), and cholesterol (Sigma, St. Louis, MO, USA) at molar ratio 3:3:2:3], along with the indicated amount of the sonosensitizer PPIX, was dissolved in a solution of chloroform:methanol 9:1. The solvent was evaporated under reduced pressure, and the lipid was redissolved in t-butanol, followed by freeze-drying. The lipid cake was hydrated with PBS, TTX solution (0.375 mg/mL PBS; Abcam, Cambridge, MA, USA), or sulforhodamine B solution (10 mg/mL PBS; Aldrich, St. Louis, MO, USA). After 10 freeze–thaw cycles, the solution was dialyzed against PBS for 48 h in a dialysis tube with a molecular mass cut-off of 1000 kDa. The dialysis media were changed with fresh PBS at least twice a day. Lipo-DMED was made following the same procedure, but with PPIX not included in the formulation and with hydration in 1 mg/mL of DMED in PBS solution.
3
Evaluating HUVEC Proliferation under Oxidative Stress
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Human umbilical vein endothelial cells (HUVECs) (1.5 × 105 cells/well) were plated in 24-well plates. After stimulation with H2O2 and/or pre-incubation with 200 μg/mL WS extracts or CS extracts, the cells were cultured for 24 h. Next, the supernatant was aspirated, and the cells were fixed with 100 μL of 10% cold trichloroacetic acid at 4 °C for 1 h, washed with distilled water five times, and air-dried. One hundred microliters of 4 mg/mL sulforhodamine B solution (Sigma Aldrich, Carlsbad, CA, USA) were added into each well and dyed for 15 min at room temperature, followed by removal of the supernatant, washing five times with 1% acetic acid, and air-drying. Afterward, 150 μL/well of Tris solution was added, and the solution was shaken at 37 °C for 20 min. The optical density value was measured at a wavelength of 540 nm under a microplate reader to calculate the cell proliferation activity.
4
Cytotoxicity Assay of Cell Lines
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Inoculation of single cells was conducted on 96-well plates at a density of 104 cells/well (for HeLa, Hep G2, fibroblast, and MCF-7 cell lines), 7.5 × 103 cells/well (for the NCI H460 line), and 5 × 104 cells/well for the Jurkat cell line. After 24 h of culture, the cell population was incubated with the probe at different concentrations for 48 h. Then, the total protein fixing of test cells was conducted with a cold solution of 50% trichloroacetic acid (Sigma, St. Louis, MO, USA) (Jurkat alone was 70%) and stained with 0.2% sulforhodamine B solution (Sigma). The results were read with an ELISA reader at two wavelengths of 492 nm and 620 nm. The experiments were triplicated and the results are presented as mean ± standard deviation.
After obtaining the optical density values at 492 nm and 620 nm (denoted OD492 and OD620):
Calculate the percentage (%) of cytotoxicity according to the following formula:
where,
After obtaining the optical density values at 492 nm and 620 nm (denoted OD492 and OD620):
Calculate the percentage (%) of cytotoxicity according to the following formula:
where,
5
Sulforhodamine B Cytotoxicity Assay
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4 × 104 cells were seeded in a 96-multiwell and stimulated with F1, F2, F3, F4, F5, F6 at concentration of 0.1, 1, 10, 0.3, 3, 30 µg/mL, respectively. After 24, 48, and 72 h the cells were fixed for 1 h at 4 °C by gently layering 1/4 volume of cold 50% (w/v) Trichloroacetic Acid (TCA Solution) on top of the growth medium, and then rinsed with water several times to remove TCA solution, serum proteins, etc. Plates were air dried and stored until use. Blank background optical density was measured in wells incubated with growth medium without cells. The 0.4% Sulforhodamine B Solution (Sigma-Aldrich Catalog Number S2902) was added in a sufficient amount to cover the culture surface area (∼50% of the culture medium volume). Cells were stained for 20–30 min and at the end of the staining period, the stain was removed, and the cells quickly rinsed with Wash Solution (1% acetic acid) until unincorporated dye was removed. The incorporated dye was then solubilized in a volume of Sulforhodamine B Assay Solubilization Solution (10 mM Tris) equal to the original volume of culture medium. Absorbance at a wavelength of 565 nm was spectrophotometrically measured.
6
Sulforhodamine B Cytotoxicity Assay
7
Cytotoxicity Evaluation of Cell-Scaffold Interactions
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For the cytotoxicity assays, MG-63 cells were seeded in six-well plates (1×104 cells/well). The cytotoxicity levels were evaluated through the contact between the cells and the scaffolds inserted in the upper chambers of the transwell plates. After 7 days, the chambers were removed, and the cells were washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) and fixed with 10% trifluoroacetic acid (1 hour at 4°C). After washing and drying, the cells were stained with 0.4% sulforhodamine B solution in 1% acetic acid (Sigma-Aldrich) for 30 minutes at room temperature. The cells were rewashed with 1% acetic acid to remove the non-bonded dye and left to dry. The bonded dye was solubilized in 1 mM Tris (Sigma-Aldrich), and the solution was stirred and transferred to a 96-well plate for absorbance evaluation in a microplate spectrophotometer (Synergy HT Multi-Detection; BioTek, Winooski, VT, USA) at 570 nm. The experiments were performed in triplicate. The data were expressed as the average with standard deviation. Statistical comparisons were carried out via one-way analysis of variance (ANOVA) and multiple comparisons Tukey’s test to determine any significant differences between groups (P<0.05).
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