SRB assays have been described in detail elsewhere [27 (link)]. SRB (Alfa-Aeser, Heysham, UK) stock solution was prepared in 1% (v/v) acetic acid at a concentration of 0.057% (v/v). A 100 µL volume of cold TCA 10% (v/v) (Trichloroacetic acid, Sigma-Aldrich, Gillingham, UK) was used to fix HEK 293 cells which were previously treated with thymol solutions and incubated at 4 °C for 1 h. The TCA was removed under a gentle stream of running tap water, then the plate was left to air dry at room temperature. The cells were stained with SRB solution (100 µL/well) for 30 min incubated at room temperature, then SRB was aspirated, and the cells were washed quickly with 1% (v/v) acetic acid to remove the excess dye. The SRB was extracted with 200 µL of 10 mM Tris base solution (Sigma-Aldrich, Gillingham, UK), having a pH of 10.5. The absorbance was measured at λ540nm and finally, the biocompatibility was calculated as the percentage of the absorbance of treated to untreated cells.
Tris base solution
Manufactured by Merck Group
Sourced in United States
Tris base solution is a buffer commonly used in biochemistry and molecular biology applications. It is an aqueous solution of tris(hydroxymethyl)aminomethane, a compound that acts as a pH regulator. The solution maintains a stable pH environment, making it suitable for various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Trichloroacetic acid, by Merck Group (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Synergy ht, by Merck Group (1 mentions)
Clear 96 well plate, by Corning (1 mentions)
Sulforhodamine b srb, by Merck Group (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Accutom 50, by Struers (1 mentions)
Synergy ht multi detection microplate reader, by Agilent Technologies (1 mentions)
Trichloroacetic acid solution, by Merck Group (1 mentions)
Acetic acid solution, by ITW Reagents (1 mentions)
Srb solution, by Merck Group (1 mentions)
6 protocols using tris base solution
1
Sulforhodamine B Biocompatibility Assay
2
Cell Viability Assay with CCK-8 and SRB
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Viable cells were counted using trypan blue staining and a haemocytometer. 2500 cells (5000 BxPC-3 cells) were seeded onto clear 96-well plates (Corning). Wells containing only media and vehicle acted as blanks. Plates were then incubated for 2–96 h. At each time-point, 10 μL of CCK-8 (Dojindo, Munich, Germany) was added to each well and plates were incubated for another 60 min at 37 °C. Following incubation, the plate was read for absorbance at 450 nm using a Synergy HT plate reader, operated by Gen 5 software (both Biotek, Whiting, Vermont, USA). Absorbance from blank wells containing only DMEM and vehicle were subtracted from each well reading. Cells were then fixed using of 100 μL 10% trichloroacetic acid at 4 °C for 1 h, washed with milliQ water and dried at 50 °C. Fixed cells were stained using 100 μL 0.057% sulforhodamine-B (SRB) (Sigma) dissolved in 1% acetic acid. Unbound dye was washed off of the cells using 1% acetic acid and plates were then dried at 50 °C. Bound dye was re-suspended in 200-μL 10-mM Tris-Base solution (Sigma) and absorbance at 540 nm was read using the Synergy HT plate reader. There were 4 replicates for each condition in every experiment.
3
Porcine Hydroxyapatite Synthesis and Characterization
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Porcine hydroxyapatite (PHA) was prepared according to our previous studies [68 (link),69 (link)]. Briefly, cancellous bone harvested from the porcine femoral epiphysis was immersed in 30% H2O2 and 75% ethanol respectively for 24 h to remove soft tissues. The bones were dissected into regular blocks (10 mm∗10 mm∗5 mm dimensions) by cut-off machines (Accutom-50, Struers, Ballerup, Denmark) and calcined at 800 °C for 2 h in a muffle furnace (SGM6812BK, Sigma Furnace Industry, China). The PHA blocks were then grinded into particles and filtered by screen with 0.2 mm and 0.5 mm hole size to obtain 0.2–0.5 mm PHA particles. Image J has been performed to measure the maximum and minimum Feret diameter of the PHA particles. For PDA coating formation, dopamine hydrochloride (Sigma, USA) was dissolved in a 0.01 M Tris-base solution (Sigma, USA) to obtain a 2 mg/mL polydopamine (PDA) solution. The PHA particles were immersed in the PDA solution for 48 h to form a PDA nano coating on PHA (PDA-PHA). Next, SEM, FTIR, XRD, and XPS were used to characterize the biomaterials.
4
Sulphorhodamine B (SRB) Assay for Cell Density
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The sulphorhodamine B (SRB) assay was used to determine cell density based on the total cellular protein content (biomass by total protein) by staining cellular proteins with SRB [50 (link),51 (link)]. As previously described for the MTT assay, 1 × 104 cells per well were seeded in each well of a 96-well tissue culture plate and left overnight in the incubator. The cells were incubated with the compounds for 24, 48, or 72 h similar to the MTT assay.
At each time point, the cells were fixed with 100 μL cold 40% (w/v) trichloroacetic acid (Sigma) solution in deionised water. The plates were kept for 1 h at 4 °C and washed five times with water. A SRB solution (0.4% SRB in 0.1% acetic acid) was prepared beforehand. The SRB solution (100 μL per well) was added, and the plates were left at room temperature for 1 h. Subsequently, the plates were rinsed with 1% acetic acid solution, flicked to remove unbound dye, and left to air-dry overnight prior to adding 100 μL of 10 mM TRIS base solution (Sigma) to each well. The plates were left on a shaker for 30 min and the optical density (OD) at 492 nm was measured in a Synergy ™ HT multi-detection microplate reader (BioTek Instruments, Inc.). The percentage change in cell growth was then calculated (OD (sample)/OD (blank) × 100). This assay was performed in triplicate.
At each time point, the cells were fixed with 100 μL cold 40% (w/v) trichloroacetic acid (Sigma) solution in deionised water. The plates were kept for 1 h at 4 °C and washed five times with water. A SRB solution (0.4% SRB in 0.1% acetic acid) was prepared beforehand. The SRB solution (100 μL per well) was added, and the plates were left at room temperature for 1 h. Subsequently, the plates were rinsed with 1% acetic acid solution, flicked to remove unbound dye, and left to air-dry overnight prior to adding 100 μL of 10 mM TRIS base solution (Sigma) to each well. The plates were left on a shaker for 30 min and the optical density (OD) at 492 nm was measured in a Synergy ™ HT multi-detection microplate reader (BioTek Instruments, Inc.). The percentage change in cell growth was then calculated (OD (sample)/OD (blank) × 100). This assay was performed in triplicate.
5
SRB Assay for Evaluating Anti-Cancer Potential
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The SRB assay was adapted from the procedure used in the NCI’s in vitro anti-cancer drug screening [29 (link),30 (link)]. Briefly, cells were plated in 96-well plates at their previously determined optimal concentrations (5.0 × 104 cells/mL for NCI-H460 and MCF-7 cells and 1.0 × 105 cells/mL for HCT-15 cells) and incubated for 24 h. Cells were then treated with five serial dilutions of T. lignosa infusion and T. lignosa decoction, ranging from 400 µg/mL to 25 µg/mL. Doxorubicin was used as a positive control (ranging from 150 nM to 9.37 nM). The effect of the solvent of the extracts (water) on the growth of the cell lines was also evaluated by treating cells with the maximum concentration of water used. Following 48 h incubation with the extract, plates were fixed by adding ice-cold 10% ice cold trichloroacetic acid (TCA) (w/v, final concentration, Panreac, Barcelona, Spain) and stained with 1% SRB (Sigma Aldrich, St. Louis, MO, USA) in 1% (v/v) acetic acid. Bound dye was solubilized by adding 10 mM Tris base solution (Sigma Aldrich) and finally the absorbance was measured at 510 nm in a microplate reader (BioTek® Synergy HT, Winooski, VT, USA). Using the SRB assay, the GI50 concentration (concentration that inhibits 50% of net cell growth) was determined for T. lignosa infusion and decoction extracts in each cell line.
6
Sulforhodamine B Assay for Cell Viability
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The sulforhodamine B (SRB) assay was used to determine cell viability. 44 After being cultured with and without the compound of interest for 18 hours, adherent cells (1 × 10 5 cells per well) were fixed using 50 µL of a trichloroacetic acid solution (50% w/v) (Sigma-Aldrich, St Louis, MO, USA) and incubated at 4 °C for 1 hour. Subsequently, the plates were washed five times with distilled water and allow to air dry. Afterwards, 100 µL of SRB solution (0.4% w/v) (Sigma-Aldrich, St Louis, MO, USA) were added to each well and incubated for 30 minutes at room temperature in the dark. After that time, the supernatant was eliminated, and the plate was washed five times with an acetic acid solution (1% v/v) (Panreac, Barcelona, Spain) and left to air dry. At the end, 100 µL of a Tris base solution ( pH 10.5, 10 mmol L -1 ) (Sigma-Aldrich, St Louis, MO, USA) was added to each well. The optical density (OD) was quantified at 510 nm using a multi-well plate reader spectrophotometer (BioTek, Bad Friedrichshall, Germany) to evaluate the cell survival.
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