Compounds were dissolved in DMSO and 3‐fold dilutions in DMSO were prepared by serial dilution. Aliquots (1 µL) of each dilution were transferred to wells of flat‐bottomed 96‐well microtiter plates, with three replicate wells for each treatment, and DMSO alone in control wells. Wells then received 100 µL of YPG broth. Cells taken from overnight cultures grown on YPD medium at 30 °C were suspended in YPG broth at a cell density of 2 × 105 cells mL‐1. Wells were inoculated with 100 µL of cell suspension and the plates were incubated at 30 °C for 72 h. Growth was assessed using a NEPHELOstar Galaxy plate reader (BMG Labtech, Cary, NC, USA) and 50% effective concentration (EC50) values for growth inhibition were determined using the calcusyn program (Biosoft, Cambridge, UK).
Calcusyn program
Manufactured by Biosoft
Sourced in United Kingdom, United States
CalcuSyn is a software program designed for data analysis and combination index calculations. It is used to analyze the effects of drug combinations on cell viability or other biological responses. The program provides mathematical models and statistical tools to evaluate the synergistic, additive, or antagonistic interactions between multiple compounds.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (2 mentions)
Celltiter glo, by Promega (2 mentions)
Nephelostar galaxy, by BMG Labtech (1 mentions)
Ribavirin, by Merck Group (1 mentions)
Telaprevir, by Selleck Chemicals (1 mentions)
Boceprevir, by ChemScene (1 mentions)
Daclatasvir, by Selleck Chemicals (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Mtt cell proliferation assay kit, by Roche (1 mentions)
96 well plate, by Corning (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Facscalibur, by BD (1 mentions)
Spss version 24, by IBM (1 mentions)
Prism v6, by GraphPad (1 mentions)
Matlab, by MathWorks (1 mentions)
Ypd agar, by BD (1 mentions)
26 protocols using calcusyn program
1
Microtiter Plate Antifungal Assay
2
Synergistic Effects of AT-101 and Gefitinib
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All statistical calculations were performed by Statistical Package for the Social Sciences (SPSS) 13.0 software (Chicago, IL, USA). Results were representative of three independent experiments unless stated otherwise. In vitro results are expressed as mean ± SD and in vivo results are expressed as mean ± SE. One-way Analysis of Variance (ANOVA) test was used to analyze significance between groups. A P value of <0.05 was considered statistical significant. The synergistic effect of AT-101 and gefitinib was assessed by the Biosoft CalcuSyn program (Ferguson, MO, USA). The combination index (CI) was used to express synergism (CI < 1), additive effect (CI = 1), or antagonism (CI > 1).
3
Cell Growth Inhibition and Colony Formation Assay
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Cell growth inhibition assay was performed as described previously [28 (link)]. For cell growth experiment, cells were treated with the RRs for 6 days. MTT assay was performed at the end of the experiment. Calculations of combination indices were done using the Calcusyn program (Biosoft, Cambridge, United Kingdom). For colony formation assay, cells were plated 1000 per well in complete media in six-well plates and allowed to adhere for 24 h. The next day cells were treated with (VN/14-1, ATRA, CGP57380, cercosporamide and 4-HPR) and RRs (10 μmol/L). After 24 h compound containing media were removed, and cells were allowed to form colonies in complete media. Approximately 2–3 weeks later the colonies were fixed, stained with 0.5% crystal violet (sigma) for 30 min and counted manually. Results represent the mean ± standard deviation of three independent experiments.
4
PDAC Cell Line Apoptosis Assay
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PDAC cell lines were seeded at a concentration of 1 × 105 cells/mL per well, in a 12-well polystyrene cell culture plate (Corning, Glendale, AZ, USA). The cells were treated at different doses of GEM (1, 10, 100, 1000 µm). After 6 days, cells were labelled with PI and the DNA content measured by flow cytometry. The dose-response curves were then plotted and the drug concentrations inducing an apoptosis of 50% (IC50) were calculated. The IC50 was calculated using the nonlinear minimum quadratic curve using the CalcuSyn program (Biosoft, Oxford, UK).
5
Synergistic Effects of Gemcitabine and Glaucarubinone
6
Synergistic Drug Combination Evaluation
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The therapeutic effect of a two‐drug combination was assessed by cell viability assays, and the combination index (CI) was calculated by the calcusyn program (Biosoft, Cambridge, UK), as previously described [9 (link)]. A CI < 1 is indicative of a synergistic effect.
7
Neuraminidase Inhibition Assay for Viral Release
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Neuraminidase inhibition assay was performed to investigate the influence of a test compound on the release of newly produced viral particles, as described previously (Shen et al., 2018 (link)). Briefly, 15 μL of A/Puerto Rico/8/1934(H1N1) virus solution were mixed with 5 μL of a test compound at graded concentrations in wells of a 96-well black plate, followed by incubation at 37°C for 30 min. Then, 30 μL of 20 μM MU-NANA 2′-(4-Methylumbelliferyl)-alpha-D -N-acetylneuraminic acid sodium salt hydrate (Sigma-Aldrich, United States), as the substrate dissolved in the MES (2-(N-Morpholino) ethanesulfonic acid buffer (32.5 mM MES and 4 mM CaCl2, pH 6.5), were added to each well, followed by incubation at 37°C for 1 h in the dark. Subsequently, 50 μL of 14 mM NaOH were added to terminate the enzymatic reaction. The fluorescence intensity of the product 4-methylumbelliferone was recorded using a microplate reader with excitation and emission wavelengths of 340 and 440 nm, respectively. The IC50 values were calculated with the Calcusyn Program (Biosoft).
8
Synergistic Effects of Oncolytic Virus and Gemcitabine
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PANC-1, SW1990 and BxPC-3 cells were dispensed in 96-well culture plates at a density of 5×103 cells/well. After attachment, cells were infected with oVV, oVV-Smac with or without gemcitabine at given concentrations and times. The medium added together with PBS was used as a blank control. The cell survival rate was evaluated by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), Medium was removed and fresh medium containing MTT (5 mg/ml) was added to each well. The cells were incubated at 37°C for 4 h, and after the supernatant was drawn off of each well carefully and an equal volume (150 µl) of DMSO was added to each well and mixed thoroughly on a concentrating table for 10 min. The absorbance of the plates was read at 595 nm with a GENios model DNA Expert Microplate Reader (Tecan Group, Ltd., Mannedorf, Switzerland). For combination index plots, CI is expressed as the log10(CI) ± 1.96 SD, and the 95% confidence intervals (CIs) are shown where estimable, with the use of the algebraic approximation algorithm of the CalcuSyn program (Biosoft, Cambridge, UK). In the present study, CI values were calculated over a scope of levels of growth inhibition (GI) from 20 to 80% of the fraction affected.
9
Evaluating Antiviral Drug Synergies
10
Statistical Analysis of Synergistic Effects
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SPSS19.0 (Munich, Germany) was performed for statistical analysis. Results were representative of minimal three independent experiments unless stated otherwise. Values were presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) test was used to analyze significance between different groups. The least significant difference (LSD) method of multiple comparisons between two groups was applied when the probability for ANOVA was statistically significant. Statistical significance was determined at a P < 0.05 level. In the analysis of additivity and synergism, the theoretical zero-interaction (exactly additive) dose-response curve for each miR-146a mimic + other agent combination was calculated by applying Bliss independence criterion [45 (link)] and was also assessed by the Biosoft CalcuSyn program (Ferguson, MO, USA). The combination index (CI) was used to express synergism (CI < 1), additive effect (CI = 1), or antagonism (CI > 1) [37 , 38 (link), 40 , 41 , 43 , 46 (link)].
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