MIA PaCa-2, PANC-1, and HPAF-II cells (3000/well) were seeded in 96-well plates. The viability of control and GSTP1 knockdown cells after 24, 48, 72, and 96 h was evaluated by adding 100 μL of CellTiter-Glo® substrate to each well containing 100 μL of media. The plates were incubated for ten min at room temperature. The endpoint luminescence was measured using Synergy H1 Hybrid multi-mode plate reader (Winooski, VT, USA) located in the Core Biology Facility, Chemistry and Molecular Biology, North Dakota State University. The gain was maintained at 135 and the integration time of 1 s using the Gen5 v2.07 software. The data represent the average ± standard deviation of three independent experiments with eight technical replicates for each treatment.
Synergy h1 hybrid
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The Synergy H1 Hybrid is a versatile laboratory instrument designed to perform a wide range of analytical tasks. It combines multiple detection technologies within a single platform, enabling users to conduct diverse experiments and analyses. The core function of the Synergy H1 Hybrid is to provide reliable and accurate data collection across a variety of applications.
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14 protocols using synergy h1 hybrid
1
Cell Viability Assay for GSTP1 Knockdown
2
Caspase-8 and Caspase-9 Activity Assay
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Caspase-8 and caspase-9 activity assays were performed according to manufacturer's instruction. Cells were seeded at a density of 25,000 cells/well in white-walled 96 well plates. After treatment with different doses of xanthohumol and incubation period of 12, 24, and 48 h, the plates were equilibrated to room temperature and then 100 μL Caspase-Glo® Reagent were added to each well. After incubation for 1 h, luminescence of each test sample was measured by Synergy H1 Hybrid.
3
Sulforhodamine B Cytotoxicity Assay
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This assay method is adapted from Houghton et al. [14 (link)]. In our study, 8,000 cells/well were seeded into a 96-well plate and incubated overnight for adherence. At the end of the incubation period, medium contained in each well was discarded and replaced by various concentrations of xanthohumol or doxorubicin and retained for 24-hour, 48-hour, and 72-hour treatment periods. At the end of these treatment periods, 50 μL of 40% trichloroacetic acid (TCA) (w/v) was added to each well. The plates were incubated for 1 hour at 4°C before discarding the supernatant. Each well was subjected to five washes with 50 μL of distilled water before being air-dried. Next, 50 μL of SRB dye (0.4% w/v in 1% acetic acid) was added to each well and incubated for 30 minutes at room temperature for the staining process. To remove the unbound dye, each well was washed with 50 μL of 1% acetic acid five times before being air-dried. Next, 100 μL Tris-base (10 mM unbuffered, pH 10.5) was added to each well. The plate was shaken at 500 rpm for 5 minutes to solubilize the remaining bound SRB dyes. Absorbance values were measured at 492 nm using Synergy H1 Hybrid. This enabled the IC50 values to be determined by plotting dose response curves using percentage of inhibition of cell proliferation against treatment concentrations.
4
Biofilm Eradication Assay with Vitexin
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Biofilm was formed as described above and were then transferred to a standard 96-well plate for MBEC assay as described previously by Wu et al. 2014 with minor modification30 (link). Briefly, biofilm was incubated overnight at 37 °C with sub-MIC doses of vitexin alone and in combination with other antibiotics. Wells were rinsed with PBS, and placed in a second 96-well plate containing vitexin alone and in combination with other antibiotics. Plate was incubated once again at 37 °C for 24 h. Viability of biofilm was then determined by measuring the turbidity at 650 nm in a 96-well plate reader (Synergy H1 Hybrid). The minimal biofilm eradication concentration (MBEC) was defined as the minimal concentration of compound required to eradicate the biofilm.
5
Caspase-3 Activity Assay of Xanthohumol
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Caspase-3 activity assay was carried out according to manufacturer's instruction. Briefly, cells seeded in 6-well plates at a density of 3.0 × 105 cells/well were treated with various concentrations of xanthohumol and different incubation time (12, 24, and 48 h). At the end of treatment, cells were harvested, washed with PBS, and lysed in the cold lysis buffer for 30 min on ice. In a 96-well plate, 200 µL of HEPES buffer, 5 μL acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin (Ac-DEVD-AMC) substrate, and 50 μL cell lysate were added to each well. The reaction mixtures were incubated for 1 h at 37°C. AMC liberated was measured using Synergy H1 Hybrid with 380 nm as excitation wavelength and 440 nm as emission wavelength.
6
Vitexin Cytotoxicity on Macrophages
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Cytotoxicity of vitexin alone and in combinations was performed with murine peritoneal macrophages42 (link) and RAW 264.7 macrophage cell line43 (link) with minor modifications. For peritoneal macrophages, BALB/c mice were induced with intra peritoneal injection of starch (0.4%), after 48 h peritoneal macrophages were collected and cell count were identified. Side by side monolayers of RAW macrophages were also take from 60% confluent culture and cell count were identified. 2 × 105 cell were seeded into each well of 96-well tissue culture plates (Eppendorf) and cultured in RPMI-1640 media supplemented with 10% FCS. Peritoneal and RAW macrophages were taken in separate 96-well plate. Cells were treated with sub-MIC doses of vitexin alone and in combination (as per the scheme of the study) and incubated in presence of 5% CO2 at 37 °C. RAW macrophages were incubated for 48 h whereas peritoneal macrophages were incubated for 4 hrs. Thereafter, the medium was replaced with fresh RPMI (without Phenol Red) containing 1 mg/mL MTT and incubated at 37 °C for 3 h. Followed by that formazan crystals were dissolved in DMSO by 20 min incubation at room temperature and absorbance was measured using an automatic plate reader (Synergy H1 Hybrid) at test wavelength of 550 nm.
7
Cytotoxicity Assay of Xanthohumol and Doxorubicin
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This assay method is adapted from Houghton.[14 (link)] 8,000 cells/well were seeded into a 96-well plate and incubated overnight for adherence. At the end of incubation, media in each well was discarded and replaced by various concentrations of xanthohumol or doxorubicin for 24, 48, and 72 h treatment periods. Treatment periods were ended by adding 50 µl of 40% cold trichloroacetic acid (TCA) acid (w/v) to each well. The plates were incubated for 1 h at 4°C before supernatant were discarded. Each well was then washed with 50 µl of distilled water for 5 times and air-dried. 50 µl of SRB (0.4% w/v in 1% acetic acid) was added to each well and incubated for 30 min at room temperature for the staining process. To remove unbound dye, each well was washed with 50 µl of 1% acetic acid for 5 times and air-dried. 100 µl Tris base (10 mM unbuffered, pH 10.5) was added to each well. To solubilize bound SRB dyes, the plate was shaken at 500 rpm for 5 min. Absorbance values were measured at 492 nm using Synergy H1 Hybrid. IC50 values were determined by plotting dose response curves using percentage of inhibition of cell proliferation against treatment concentrations.
8
Amyloid Fibril Formation Kinetics of CarD
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Native tagless CarD (5, 10 and 100 μM), CarDHis (100 μM) and CarDtr (100 μM) were mixed with 10-fold molar excess of ThT and incubated for 15 min at 37 °C. Seeding experiments were performed where 5% and 10% seeds were transferred from the 100 μM CarD sample incubated at 37 °C for 5 h to 5 μM CarD. The ThT fluorescence intensity was measured using excitation wavelength of 440 nm and emission wavelength of 482 nm. The fluorescence measurements were recorded using Synergy H1 Hybrid multi-mode microplate reader. The kinetics for the formation of amyloid fibrils by CarD and CarDHis were recorded for a period of 24 h. The microplate reader was set at constant temperature of 37 °C and readings were acquired every 2 min with continuous orbital shaking between reads over a period of 24 h. The top of the each well was sealed using adhesive tape to minimize the rate of evaporation.
9
Yeast Growth Kinetics at Low pH
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We first streaked the glycerol stock of the selected strains (Fig. 1a ) on the yeast peptone dextrose (YPD) plate for growing overnight at 30°C. A single colony was picked up from the plate and inoculated into 2 mL yeast nitrogen base (YNB) broth (with amino acids and 2% glucose) at ~pH 5.5 for growing overnight at 30°C with constant shaking at 250 rpm on the platform shaker. The 2 mL seed culture was pelleted and diluted in fresh YNB broth at ~pH 5.5 with an OD600 of 1.5 for growing at 30°C with constant shaking at 250 rpm on the platform shaker for 2 h. After 2 h, the culture was pelleted and then diluted to an OD600 of 0.1 in pH 1.5 YNB broth adjusted by HCl. Three replicates of 200 µL cultures from each strain were added to the plate wells, and then OD600 was measured every 30 min for 48 h at 30°C with constant shaking in the Synergy H1 Hybrid multimode microplate reader.
10
Quantifying CD73 Enzymatic Activity
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CD73 activity was evaluated using the malachite green assay as previously described (17 (link)). Briefly, 5 μg of total cell lysate prepared from transfected cells in 200 μl of CD73 lysis buffer (10 mM Tris–HCl pH 7.5, 0.1% Nonidet P-40) was incubated at 37 °C for 10 min, followed by incubation with 2 mM AMP (Oriental Yeast Co, Ltd) at 37 °C for 30 min. Fifty microliter of the reaction solution was mixed with 100 μl of Biomol Green (Enzo Life Sciences) and incubated for 10 min at RT. The amount of inorganic phosphate released from AMP was quantified by measuring the absorbance at 595 nm using a Synergy H1 Hybrid multimode microplate reader. A standard curve was generated using 0 to 200 μM Na2HPO4 solutions dissolved in CD73 lysis buffer.
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