Calcusyn software version 2

Manufactured by Biosoft

Sourced in United Kingdom, United States

CalcuSyn software version 2.0 is a computer program designed for data analysis and curve fitting. It is used for the quantification of dose-effect relationships, including drug combinations.

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Lab products found in correlation

Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Spss software, by IBM (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Cellquest pro software, by BD (1 mentions) Modfit lt software, by Verity Software House (1 mentions) Prism 5, by GraphPad (1 mentions) Rt250, by Philips (1 mentions) Rstudio software, by Posit (1 mentions) Alamar blue, by Thermo Fisher Scientific (1 mentions) Lab tek, by Thermo Fisher Scientific (1 mentions) Gen5 microplate reader, by Agilent Technologies (1 mentions) Calcusyn software, by Biosoft (1 mentions) Celltiter glo luminescent assay, by Promega (1 mentions) Synergy htx multi mode microplate reader, by Agilent Technologies (1 mentions) Celltiter 96 cell proliferation assay kit, by Promega (1 mentions) Spss statistics v22, by IBM (1 mentions) Prism software, by GraphPad (1 mentions) Spss software 21, by IBM (1 mentions) Spss for windows version 21, by IBM (1 mentions) Sigmaplot software 12, by Grafiti LLC (1 mentions)

61 protocols using calcusyn software version 2

Synergistic Drug Interactions: Modeling and Quantification

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The type of interaction derived from MTF/PyP and PMM/PyP when they are in binary combination was described by the median-effect/combination index (CI)-isobologram equation [33 (link),34 (link)]. The CI < 1, = 1, and > 1 shows synergism, additive and antagonism effect of the combination, respectively. These interactions were analyzed with CalcuSyn software version 2.1. (Biosoft, Cambridge, UK).

Melcón-Fernández E., Galli G., Balaña-Fouce R., García-Fernández N., Martínez-Valladares M., Reguera R.M., García-Estrada C, & Pérez-Pertejo Y. (2024). In Vitro and Ex Vivo Synergistic Effect of Pyrvinium Pamoate Combined with Miltefosine and Paromomycin against Leishmania. Tropical Medicine and Infectious Disease, 9(2), 30.

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Synergistic Analysis of Bortezomib and Daunorubicin

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Statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). Student's t-test was used to compare differences between two groups. P<0.05 was considered to indicate a statistically significant difference. Synergistic interactions between bortezomib and daunorubicin were analyzed by the Chou-Talalay method using CalcuSyn software version 2.1 (Biosoft, Cambridge, UK) (13 (link)). A combination index (CI) of <1, equal to 1, and >1 indicate synergistic, additive and antagonistic effects, respectively.

Du X., Tong J., Lu H., He C., Du S., Jia P., Zhao W., Xu H., Li J., Shen Z., Wu Y., Tong J, & Zhou L. (2017). Combination of bortezomib and daunorubicin in the induction of apoptosis in T-cell acute lymphoblastic leukemia. Molecular Medicine Reports, 16(1), 101-108.

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Isobologram Analysis of Mycotoxin Interactions

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The isobologram analysis (Chou-Talalay model) was used to determine the type of interaction (synergism or antagonism effect) that occurs when mycotoxins studied were in combination. Chou-Talalay’s model allows characterizing the interactions induced by combinations of mycotoxins, but no mechanisms associated can be elucidated. Combination effects are possible to analyze by the median-effect/combination index (CI)-isobologram equation by Chou [42 (link)] and Chou and Talalay [7 (link)]. The model involves plotting the dose–effect curves for each mycotoxin and its combinations in multiple diluted concentrations. There are several parameters included in the equation as Dm (the median-effect dose), fa (fraction affected by concentration), and m (coefficient signifying the shape of the dose–effect relationship) [7 (link)]. Therefore, both the potency (Dm) and shape (m) parameters are crucial for the evaluation.
The term combination index (CI) is included in Chou and Talalay’s [42 (link)] method. According to the CI synergism, additive, and antagonism are, respectively, correlated as follows: CI < 1, =1, and >1. CalcuSyn software version 2.1. (Biosoft, Cambridge, UK, 1996–2007) was used to study the types of interactions assessed by isobologram analysis. It was decided to report CI₂₅, CI₅₀, CI₇₅, and CI₉₀ doses to produce toxicity at 25%, 50%, 75%, and 90%, respectively.

Juan-García A., Tolosa J., Juan C, & Ruiz M.J. (2019). Cytotoxicity, Genotoxicity and Disturbance of Cell Cycle in HepG2 Cells Exposed to OTA and BEA: Single and Combined Actions. Toxins, 11(6), 341.

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Synergistic Drug Combination Analysis

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The synergistic effect was analyzed by the multiple drug-effect equation and quantified by the combination index (CI) using CalcuSyn software version 2.1 (Biosoft, Cambridge, UK). CI values between 0.9–1.1 indicates an additive effect; values between 0.7–0.9 indicated a moderate synergism; values lower than 0.7 indicated a strong synergism; and antagonism is represented by CI values higher than 1.1.

Krayem M., Sabbah M., Najem A., Wouters A., Lardon F., Simon S., Sales F., Journe F., Awada A., Ghanem G.E, & Van Gestel D. (2019). The Benefit of Reactivating p53 under MAPK Inhibition on the Efficacy of Radiotherapy in Melanoma. Cancers, 11(8), 1093.

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Detailed Statistical Analysis Protocol

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Statistical analysis was performed using Graph Pad Prism statistical software version 4.03 (La Jolla, CA, USA) and RStudio software version 1.0.153 (Boston, MA, USA) Lattice package [48 , 49 ]. Test for normality, F-test for equal variance, and appropriate t-test were used for the first two animal studies, and for analysis of western blot results. For the third animal study (the dose escalation study) we used one-way ANOVA and Tukey post-hoc test analysis. IC₅₀ values were determined using CalcuSyn software version 2.1 (Biosoft, Cambridge, England). Heat maps were constructed using RStudio software version 1.0.153 (Boston, MA, USA) gplot package [49 , 50 ].

Mudd A.M., Gu T., Munagala R., Jeyabalan J., Fraig M., Egilmez N.K, & Gupta R.C. (2021). Berry anthocyanidins inhibit intestinal polyps and colon tumors by modulation of Src, EGFR and the colon inflammatory environment. Oncoscience, 8, 120-133.

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Synergistic Apoptosis Induction: Flow Cytometry Protocol

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Percentage of apoptotic cells was determined using Annexin-V/Propidium Iodide (PI) staining following the manufacturer's instructions (Becton Dickinson, Franklin Lakes, NJ, CA, USA) and analyzed employing a Becton Dickinson FACSCalibur flow cytometer and CellQuest Pro software. Synergy of drug interactions was calculated using the fixed ratio/combination index method [42 (link)]. Apoptotic cells were measured by Annexin-V/PI staining and combination index (CI) calculated using CalcuSyn software Version 2.1 (Biosoft, Cambridge, UK). CI with values < 1.0 indicate a synergistic interaction of both drugs in the combination and CI values < 0.5 indicate strong synergy. For cell cycle analysis, cells were stained with PI and analyzed by flow cytometry. Cell cycle was fitted to viable cells using ModFit LT software (Verity Software House). Changes in mitochondrial membrane potential (ΔMOMP) were stained with the membrane determined by staining cells with JC-1 as described by the manufacturer (Molecular Probes, Eugene, OR, USA) followed by flow cytometry analysis.

Heinemann A., Cullinane C., De Paoli-Iseppi R., Wilmott J.S., Gunatilake D., Madore J., Strbenac D., Yang J.Y., Gowrishankar K., Tiffen J.C., Prinjha R.K., Smithers N., McArthur G.A., Hersey P, & Gallagher S.J. (2015). Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and suppresses AKT and YAP signaling. Oncotarget, 6(25), 21507-21521.

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Synergistic Efficacy of Drug Combination

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Data from MTT assay (triplicate experiments independently repeated at least two times) were averaged and statistically analyzed by unpaired t-test using Prism 5.0 (GraphPad). A p-value less than 0.05 was considered significant. To investigate the synergistic efficacy of the drug combination, the combination index (CI) was determined according to the Chou-Talalay method using CalcuSyn software version 2.1 (Biosoft) [23 (link)].

Park H.K., Lee J.E., Lim J., Jo D.E., Park S.A., Suh P.G, & Kang B.H. (2014). Combination treatment with doxorubicin and gamitrinib synergistically augments anticancer activity through enhanced activation of Bim. BMC Cancer, 14, 431.

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Synergistic Combination Therapy Evaluation

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Statistical analyses were examined using Student’s t-test. P-values <0.05 were considered statistically significant. The interactions between SHK and VER-155008 was analyzed by Chou’s combination index (CI) using CalcuSyn software Version 2.1 (Biosoft, Cambridge, UK) to determine whether the combination was additive or synergistic (36 (link)).

WADA N., KAWANO Y., FUJIWARA S., KIKUKAWA Y., OKUNO Y., TASAKI M., UEDA M., ANDO Y., YOSHINAGA K., RI M., IIDA S., NAKASHIMA T., SHIOTSU Y., MITSUYA H, & HATA H. (2014). Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells. International Journal of Oncology, 46(3), 963-972.

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Combination Treatment of ATO and Radiation

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Cells were seeded into 12-well plates at a density of 300 cells/well. Twenty four hours after seeding, cells were left untreated or were treated with increasing doses of ATO, irradiation (IR) or the combination of both. Irradiation was performed using a 250 kV deep x-ray unit (Philips RT250) and single doses up to 6 Gy were applied. Non-irradiated cultures were processed along with irradiated cultures. If not stated otherwise, cells were incubated with ATO 2 hs before irradiation for combined treatment. Cells were then incubated for up to 14 days. At the end of the experiments colonies were fixed, stained using 10% Giemsa stain solution and colonies containing >50 cells were counted. Plating efficiency and survival fractions for given treatments were calculated on the basis of survival of non-treated cells. All samples were done in triplicates and at least three independent experiments were carried out. The median inhibitory concentration (IC50) of ATO and the combination index (CI) for the combined treatment with ATO and IR were calculated using the CalcuSyn software version 2.1 (Biosoft, Cambridge, UK).

Boyko-Fabian M., Niehr F., Distel L., Budach V, & Tinhofer I. (2014). Increased Growth-Inhibitory and Cytotoxic Activity of Arsenic Trioxide in Head and Neck Carcinoma Cells with Functional p53 Deficiency and Resistance to EGFR Blockade. PLoS ONE, 9(6), e98867.

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Combination Drug Synergy Assay

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In the in vitro combination studies, cells were seeded in 96-well plates (2000 cells/well) and incubated at 37 °C for 24 h before addition. Then cells were treated with RO4929097, BKM120 and BEZ235. Cell viability was quantified with the CellTiter-Blue assay. Drug synergy was analyzed by calculating the combination index (CI) as a measure of the interaction between two drugs. The Combination Index (CI) was calculated according to the median-effect principle of the Chou and Talalay method, using the CalcuSyn software, version 2.1 (BioSoft, Great Shelford, Cambridge, UK). CI values are generated over a range of Fa levels, from 0.05–0.90 (5–90% growth inhibition). A CI of 1 indicates an additive effect between two agents, whereas a CI of <1 or >1 indicates synergism or antagonism, respectively [32 (link)].

Saito N., Hirai N., Aoki K., Suzuki R., Fujita S., Nakayama H., Hayashi M., Ito K., Sakurai T, & Iwabuchi S. (2019). The Oncogene Addiction Switch from NOTCH to PI3K Requires Simultaneous Targeting of NOTCH and PI3K Pathway Inhibition in Glioblastoma. Cancers, 11(1), 121.

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