MDA-MB-231 or ZR-75-1 cells were seeded onto tissue-culture treated 96-well plates (Costar #3595, Corning, Inc., NY; n=3000 per well) in 100 μL of complete media, as described in the cell culture section, and incubated overnight at 37°C. TFP and TRA-8 were diluted in culture medium from stock solutions immediately before use. Cells were treated with 0.5 μg/mL of TRA-8 for 3 hours, or 10 μM of TFP only for 30 minutes or 10 μM of TFP for 30 minutes followed by 0.5 μg/mL of TRA-8 for 3 hours, which were the same treatment conditions as those for the characterization of the effect of TFP on DR5-mediated caspase cleavage as described above. Cell viability was assessed by the measurement of cellular ATP levels using the ATPLite luminescence-based assay (Perkin Elmer Waltham, MA), following the manufacturer’s recommended protocol. Cell viability assay was repeated six times.
Costar 3595
Manufactured by Corning
Sourced in United States
The Costar 3595 is a 96-well cell culture plate designed for use in various laboratory applications. It features a flat bottom and clear polystyrene construction, providing a suitable surface for cell growth and observation. The product dimensions and well configuration adhere to standard microplate standards.
Automatically generated - may contain errors
Lab products found in correlation
Cytation 3 microplate reader, by Agilent Technologies (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Cultilab (2 mentions)
Atplite luminescence based assay, by PerkinElmer (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Triton x 100, by Serva Electrophoresis (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Dutscher (1 mentions)
Ae2000, by Motic (1 mentions)
Inverted microscope, by Motic (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Sunrise absorbance reader, by Tecan (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Phenazine methosulfate, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
37 protocols using costar 3595
1
Cytotoxic Effects of TRA-8 and TFP
2
Antioxidant Activity Assay using DPPH
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This assay was performed as described previously [10 (link)] but with some modifications. Fifty microliters of each sample were mixed with 200 µL of 450 µmol/L DPPH solution, and then, the mixture was incubated at 30 °C for 20 min. Subsequently, each mixture was centrifuged at 1500 rpm for 5 min at 15 °C. Then, 150 µL of each supernatant was placed per well in a 96-well plate (Costar® 3595, Corning Incorporated, New York, NY, USA). The absorbance response was monitored at 517 nm for each sample in wells using the Cytation™ 3 microplate reader and Gen5™ software version 2.06 (Biotek Instruments Inc., Winooski, VT, USA). The results of this assay were expressed as the percentage inhibition of the radical or µg of ascorbic acid equivalents (AAE)/mL of sample. The percentage of inhibition against the DPPH radical was calculated using the absorbance values of the blank (distilled water) and experimental samples. The value expressed in µg AAE/mL of samples was obtained by interpolating the response of diluted samples into ascorbic acid calibration curves ranging from 6.0 to 120.0 µg/mL. For these experiments, ascorbic acid and distilled water were included as the positive and negative controls.
3
Antibacterial Efficacy of LysCP28 on Duck Meat
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Duck breast meat was cut aseptically into 2 cm × 2 cm squares (about 1 g) and disinfected with UV to minimize contamination by spoilage microorganisms. C. perfringens ATCC 13124 was grown overnight to reach an OD600 of 1.0. For these assays, sterilized duck squares were immersed in a suspension containing C. perfringens overnight cultures diluted with PBS (1:1) for 15 min at an inoculum volume of approximately 1 × 103–1 × 104 CFU/g [30 (link)]. After that, the duck squares were removed and placed in 24-well plates (Costar® #3595, Corning Incorporated, Corning, NY, USA). Finally, 0.1 mL of LysCP28 (5–500 µg/mL) was spotted onto the duck squares, and the same capacity of PBS was dropped onto inoculated duck squares as a negative control by 0.1 mL pipette accurately. Three replicates were prepared and measured for each treatment. The 24-well plates were stored immediately in a Ziploc bag for incubation at 4 °C for 3 d. The duck squares were transferred to test tubes at 0, 1, 12, 24, 48, and 72 h, 10 mL PBS solution was added, and the squares were shaken at room temperature for 30 min. The collected liquid portion was transferred to a sterile tube and centrifuged at 3000× g for 10 min. Cells were harvested and then enumerated on TSC and SFP media. The number of bacteria were estimated by the dilution gradient multiplied by the number of bacteria harvested.
4
Antimicrobial Effects on P. aeruginosa Biofilm
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An overnight culture of P. aeruginosa was cultured in MHB at 37 °C, and then diluted to 1 × 108 CFU/mL in fresh MHB. Hundred μl of bacterial suspension, containing 0–20 μM of HDP, was added to each well of 96-well (flat bottom) polystyrene microtiter plates (Costar® 3595; Corning Incorporated). After the incubation, the medium was aspirated gently and the wells of the plates were washed three times with 150 μl phosphate buffered saline (PBS) to remove unattached bacteria.
For determination of the biofilm biomass, crystal violet (CV) staining was used. Hundred μl of 99% methanol was added per well for 10 min for fixation, then aspirated, and plates were allowed to dry. Wells were stained with 100 μl 0.04% CV (Klinipath, Duiven, The Netherlands) for 20 min. Excess colorant was eliminated by three successive washes with sterile PBS. Finally, bound CV was solubilized with 33% acetic acid and optical absorbance was determined at 630 nm, using a microtiter plate reader (FLUO star Omega, BMG LABTECH, Germany). In order to determine the number of viable bacteria, biofilms were solubilized with 200 μl 0.1% Triton X-100 (Serva, Heidelberg, Germany). Bacterial cells were serially diluted and spread on agar plates and incubated o/n at 37 °C and subsequently counted.
For determination of the biofilm biomass, crystal violet (CV) staining was used. Hundred μl of 99% methanol was added per well for 10 min for fixation, then aspirated, and plates were allowed to dry. Wells were stained with 100 μl 0.04% CV (Klinipath, Duiven, The Netherlands) for 20 min. Excess colorant was eliminated by three successive washes with sterile PBS. Finally, bound CV was solubilized with 33% acetic acid and optical absorbance was determined at 630 nm, using a microtiter plate reader (FLUO star Omega, BMG LABTECH, Germany). In order to determine the number of viable bacteria, biofilms were solubilized with 200 μl 0.1% Triton X-100 (Serva, Heidelberg, Germany). Bacterial cells were serially diluted and spread on agar plates and incubated o/n at 37 °C and subsequently counted.
5
Immortalized Dental Papilla Cell Culture
6
Vaginal Epithelial Cell Infection Assay
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Vaginal epithelial cell infections were performed using 96-well plates (Costar 3595, Corning, New York, NY, USA). A-431 cells (5 × 105 cells/mL, 200 μL per well) were seeded and then incubated overnight at 37 °C with 5% CO2. Before being infected, the epithelial cell monolayer was washed with Phosphate-Buffered Saline (PBS, Dutscher, Bernolsheim, France) and kept at room temperature. Then epithelial cells were infected with C. albicans, in the presence or not of RBG, by using a Multiplicity of Infection (MOI) of 1:1, namely 5 × 105 CFU/mL.
7
In vitro Nematicidal Bioassay of Secretomes
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In vitro nematicidal activity bioassays employing M. incognita J2 larvae in aqueous suspension were performed according to the methodology of Ala et al. (2020) (link) with some modifications. Twenty microliters of secretomes obtained from submerged cultures were added to 96-well flat-bottom sterile polystyrene microplates (Corning® Costar® 3,595) containing 40–60 J2 larvae suspended in 200 μL of sterile tap water per well. Subsequently, the plates were sealed with parafilm and incubated at 25°C in the dark for 48 h, and motile and immotile larvae (considered death) were counted at 10X amplification (MOTIC, AE 2000, inverted microscope).
All experiments were performed considering three biological replicates (with three technical repeats each). Nematicidal activity was estimated according toEquation (1) .
All experiments were performed considering three biological replicates (with three technical repeats each). Nematicidal activity was estimated according to
8
Antibacterial Activity Quantification
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Antibacterial testing was performed using Escherichia coli MG1655 (ATCC 47076), Pseudomonas aeruginosa PAO1 (ATCC 15692), and Staphylococcus aureus Rosenbach (ATCC 12600). The strains were cultured overnight at 37 °C in liquid growth medium (Luria Bertani, LB) (Scharlab, Spain) for E. coli and P. aeruginosa and tryptic soy broth (TSB) (Scharlab, Spain) for S. aureus. Bacterial samples were ultrasonically dispersed in growth medium for 30 s (A = 18%, W = 250 W, on:off = 2:1 s) to form 2 mg/ml stocks, which were further diluted to the tested concentrations. Microtiter plate wells (96-well assay plate, tissue culture-treated polystyrene; Costar 3595, Corning Inc., Corning, NY) were inoculated with 100 μl of bacteria (OD550 = 0.05) and 100 μl of the specific serial dilutions of the tested samples. Controls included growth medium and bacteria without treatment. Incubation was performed in an Infinite M200 Pro multimode microplate reader (Tecan) at 37 °C for 14 h with continuous orbital shaking. Bacterial growth was assessed by measuring optical density at 550 nm every 15 min. All concentrations were tested repeatedly in three replicates (n = 3).
9
Fitness Assay for Inhibitory Substrate Tolerance
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Cell growth when exposed to varying concentrations of the inhibitory substrate was used to measure the fitness of the strains. Prior to the assays, cells we pre-adapted with overnight incubations in undiluted inhibitory substrate at 30 °C. For fitness assays, adapted cells were washed 3× in 10 mM sodium citrate pH 5.5 and inoculated to a final concentration of ~ 2 × 105 into YNB 1% glucose supplemented with varying concentrations (0–85%) of the inhibitory substrate in 96-well plates (Costar 3595, Corning). Plates were incubated at 30 °C with shaking in a Tecan Sunrise absorbance reader, measuring absorbance at 595 nm every 20 min. Because inhibitors may affect growth rate, lag time, maximum cell density or any combination thereof, we used the area under the growth curves as a measure of fitness. The lignocellulosic inhibitory substrate was kindly supplied by Tembec (Témiscaming, Québec) and AV Cell (Atholville, New Brunswick). The pH of the substrate was adjusted to 5.5 with 10 M NaOH prior to use in culture medium.
10
Halophytophthora Mycelial Growth Under Abiotic Factors
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Mycelial growth of the Halophytophthora isolates under various combinations of temperatures, pHs and salinities were measured based on a microtitre plate (Co-Star 3595, Corning, Maine, USA) method designed by Langvad (1999 ). A medium containing 4 g/L glucose, 4 g/L yeast extract and 4 g/L peptone was used as the growth medium and 180 μl of this medium adjusted to various pHs (6, 7, 8) and salinities (4 ‰, 8 ‰, 16 ‰, 32 ‰ made by sea salt) were dispensed into wells of the microtitre plate. Acidity/alkalinity of the medium was adjusted using hydrochloric acid (Taiwan Green Version Technology Ltd., New Taipei City, Taiwan) and sodium hydroxide (Panrea, Barcelona, Spain). The spore suspensions (20 μl) were added to the wells and mixed briefly. For each treatment, four replicates (wells) were done. The inoculated plates were incubated at 15°C, 25°C and 37°C. A set of control wells for each treatment, i.e. the growth medium with no spores, was also prepared. Absorbance of the wells of the microtitre plates was measured daily at 630 nm using the multi-detection microplate readers (Synergy HT, BioTek) for 9 days. Growth of the isolates was represented by subtraction of the absorbance of the inoculated wells to that of the control wells.
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