BSCP-1 was tested for its cytotoxic activity against the HGC27 human gastric carcinoma cancer cell line using the CellTiter-Glo™ luminescent cell viability assay (Promega) [20 (link)]. The HGC27 cell line was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The cells were maintained at 37°C in a humidified environment containing 5% CO2. Cell viability was determined using the CellTiter-Glo™ assay. Briefly, the cancer cells were seeded onto 384-well plates at an initial density of 2000 cells/well in 40 μL of medium. Then, the cells were treated with the compounds at varying concentrations. The dose-effect curve of doxorubicin cytotoxicity against the BEL-7402 cells was used as a positive control, and 0.5% DMSO was used as a negative control. After incubation for 72 h, 30 μL/well of CellTiter-Glo™ reagent was added, and the relative light unit (RLU) values were read on a PHERAstarplus microplate reader (BMG Labtech, Cary, NC) after incubation for 10 min at room temperature: cell growth inhibition % = 100% × [1-RLUtreatment/RLUnegative control]. The IC50 values were calculated from the curves generated by plotting cell growth inhibition versus the test concentrations using GraphPad Prism 6.0 software [21 (link)].
Pherastar plus microplate reader
Manufactured by BMG Labtech
Sourced in Germany
The PHERAstar Plus is a high-performance microplate reader that provides accurate and reliable measurement of fluorescence, luminescence, and absorbance in a wide range of applications. It features advanced optics and detection systems to ensure precise and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Prism 6, by GraphPad (1 mentions)
Celltiter glo, by Promega (1 mentions)
Bright glo luciferase substrate, by Promega (1 mentions)
Sigmaplot 13, by Grafiti LLC (1 mentions)
Celltiter glo ctg reagent, by Promega (1 mentions)
Ls6500 scintillation counter, by Beckman Coulter (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Picogreen reagent, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
8 protocols using pherastar plus microplate reader
1
Cytotoxicity Evaluation of BSCP-1 against HGC27 Gastric Cancer
2
Virus Neutralization Assay Protocol
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Serum and plasma samples or monoclonal antibodies (sterile filtered, 0.22 μM) were titrated 2-fold or 3-fold down a 96-well flat-bottom white plate (Thermo) containing complete DMEM (leaving wells without sample for virus and cell only controls) and then incubated with a 200 TCID50 dilution of PV for 1 hr at 37°C. Serum and plasma samples were diluted prior to titration to have a starting dilution of 1:100 (or 1:75 or 1:50) after the addition of PV. mAbs were used at different starting concentrations (0.5 μg/mL - 10 μg/mL) depending on their potency. HeLa TZM-bl reporter cells (1×104 cells/well) containing 25 μg/mL DEAE dextran were added and incubated for 48 hrs in a 37°C incubator with 5% CO2. Media was removed from each well prior to addition of 100 μL Bright-Glo™ luciferase substrate (Promega) diluted 1:20 in 1× lysis buffer. The luciferase activity in RLU was measured using a PheraStar Plus microplate reader (BMG Labtech). Serum and plasma 50% inhibitory dilution (ID50) values were calculated from sigmoidal dose-response curves using GraphPad Prism software. Neutralisation scores were calculated from log-transformed titers as in40 (link), using the equation Y = log3 (dilution/100) + 1.
3
Kinetic Characterization of GUS Enzymes
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p-Nitrophenyl glucuronide (PNPG) was purchased as a solid and dissolved in water at a concentration of 100 mM. Reactions were conducted in 96-well, black, clear-bottom assay plates (Costar) at 37°C. Reaction mixtures consisted of PNPG (at various concentrations) and GUS enzyme (at various concentrations) diluted in assay buffer (50 mM HEPES and 50 mM NaCl for pH ≥6.0 or 50 mM sodium acetate and 50 mM NaCl for pH <6.0). To determine the optimal pH for BoGUS L2, BuGUS L2, and BfGUS mini-loop 1, the assay described above was conducted at 800 μM PNPG for BuGUS L2 and BfGUS mini-loop 1 and 1,500 μM PNPG for BoGUS in the appropriate assay buffer where the pH ranged from 4.0 to 7.4. Reactions were quenched with 0.2 M sodium carbonate, and the product formation was measured over time via absorbance at 410 nm using a PHERAstar Plus microplate reader (BMG Labtech). Upon determining the optimal pH for each enzyme, velocities were determined for multiple concentrations of substrate and enzyme at each enzyme’s optimal pH, and the Michaelis-Menten kinetics module in SigmaPlot 13 (Systat Software, Inc.) was used to calculate Km, kcat, and kcat/Km.
4
Cell Viability Quantification via CellTiter-Glo 3D
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After the cells were grown for the desired amount of time, 100 μL of the CellTiter-Glo 3D reagent was added to each well of the 2D and 3D cultures. The plates were placed on a plate shaker for 5 min, followed by incubation for 30 min at room temperature. For 2D-cultured cells, 100 μL of the solution from each well of the 48-well plate was transferred to a white, clear-bottom 96-well plate after incubation, followed by the addition of the CellTiter-Glo 3D reagent. The luminescence was measured using the PHERAstar Plus microplate reader (BMG Labtech, Offenburg, Germany).
5
Measuring Cell ATP Levels
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Cell ATP levels were measured as described11 (link). Assay plates were removed from the incubator and allowed to reach room temperature. CellTiter-Glo (CTG) reagent (Promega) was added (20 µl well−1), with gentle agitation for 30 min. Luminescence as a measurement of cellular ATP levels was read on a PHERAstar Plus microplate reader (BMG Labtech). Results were normalised to those of untreated control cells (no death signal, no MAP4K4 inhibitor) and to 100% cell death (addition of 0.1% Triton X-100, 2 h before CTG). Normalised values were plotted against the log concentration of the death inducer or inhibitor.
6
Enzymatic Activity Assay for CED-3 and HCASP3
7
Lipid Kinase Assay Protocol
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Lipid kinase assays were performed as described previously [13 (link)]. Dipalmitoyl-PI5P (DiC16–PI5P) was purchased from Echelon Biosciences and after drying down in Eppendorf tubes was sonicated for 3 × 30 s in a Decon Ultrasonics sonicating bath. This lipid substrate (6 μM PI5P) and recombinant lipid kinase were added to the reaction mixture (200 μl of final volume) with 10 μCi [γ-32P]ATP and incubated at 30°C for 10–60 min. Lipids were extracted using an acidic phase-separation [19 (link)] and separated by 1D thin layer chromatography (2.8:4:1:0.6 chloroform:methanol:water:ammonia). Radiolabelled PtdIns(4,5)P2 spots were detected by autoradiography, extracted and counted with Ultima Gold XR scintillant (Packard) on a LS6500 scintillation counter (Beckman Coulter). Intrinsic ATPase activities of the enzymes were determined using the Transcreener ADP2 fluorescence polarization method (BellBrook Labs). A range of enzyme concentrations was assayed with ATP substrate (100 μM ATP, 60 min incubation at 22°C) and polarization units (mP) were read using a PHERAstar Plus microplate reader (BMG Labtech). Experimental values were interpolated from an ADP/ATP utilization standard curve and plotted using non-linear regression analysis with Prism 5.
8
FOXK2 Chromatin Immunoprecipitation Assay
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The cell lines were transfected with pCMV5-FOXK2WT, pCMV5-FOXK2 K527/633 R and the empty vector pCMV5 for 24 h, after which FOXK2-overexpressing cells were collected for the ChIP assay, as previously described9 (link). For the immunoprecipitation, 4 µg of either IgG (P0447, Dako; Cambridgeshire, UK) and FOXK2 (ab5298; Abcam; Cambridge, Massachussetts, USA) antibodies were added to the precleared samples. DNA quantification was performed using the picogreen reagent (Life Technologies) and fluorescence was measured in a PHERAstarPlus microplate reader (BMG Labtech, Ortenberg, Germany). For PCR reaction, we used 2.5 µL DNA from each sample, 0.5 µL of mix of primers (50 nM final concentration), 5 µL SYBR green master mix (Applied Biosystems) and 2 µL DEPC-treated water per well. The reaction was run in 7900 HT Fast Real-time PCR System(Applied Biosystems) and the cycling program was 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 95 °C for 30 s, followed by a dissociation step. The pair of primers used for ChIP was: FOXO3-F, 5′-ACCAACATCTTCGCCGTTC-3′ and FOXO3-R, 5′-GGTGTCCGGTTCCCTGTTAG-3′. All experiments were done in triplicates and results were normalized to the IgG antibody.
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