The β-galactosidase assay was performed using 96-well microplates by referring to a reported work (Schaefer et al., 2016 (link)). The obgE:: pVIK111 and deaD:: pVIK111 strains were cultured in 10 ml LB at 25°C with shaking at 200 RPM. When the OD600 reached the value of 0.2, 0.5, and 1.0, 20 μl of the culture was collected and mixed with 80 μl permeabilization solution (100 mM Na2HPO4, 20 mM KCl, 2 mM MgSO4, 0.04% sodium deoxycholate, 5 mM β-mercaptoethanol, and 1 mg/ml lysozyme) in a 96-well microplate (NuncTM MicroWellTM, Thermo Scientific, Waltham, MA, United States). Permeabilizing samples were stored at 4°C until all the samples were prepared, and then, 25 μl of permeabilized samples were mixed with 150 μl of substrate solution (60 mM Na2HPO4, 40 mM NaH2PO4, 1 mg/ml ONPG, and 5 mM β-mercaptoethanol) in a 96-well microplate (NuncTM MicroWellTM, Thermo Scientific, Waltham, MA, United States) and mixed well before loading the plate without its lid into the TECAN SparkTM 10M multimode microplate reader. The OD420 absorbance was measured every 5 min for a total of 80 min incubation at 37°C. The settings for the OD420 absorbance measurement were 25 flashes and 120-ms wait time between wells. Biological triplicates were analyzed for all strains.
Nunc microwell
Manufactured by Thermo Fisher Scientific
Sourced in United States, Belgium
The Nunc MicroWell is a microplate designed for a variety of laboratory applications. It features a 96-well format with a flat bottom well shape. The product is made of polystyrene material.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Spark 10m plate reader, by Tecan (3 mentions)
Spectramax 340pc, by Molecular Devices (2 mentions)
Ivis spectrum system, by PerkinElmer (2 mentions)
Safire microplate reader, by Tecan (1 mentions)
Nunc f96 microwell, by Thermo Fisher Scientific (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Sterile peptone water, by Thermo Fisher Scientific (1 mentions)
Amies transport medium, by Deltalab (1 mentions)
Newborn calf serum, by Thermo Fisher Scientific (1 mentions)
Lactalbumin hydrolysate, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Ckx31, by Olympus (1 mentions)
Co2 incubator, by Thermo Fisher Scientific (1 mentions)
Ir p1, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Luciferin ef, by Promega (1 mentions)
31 protocols using nunc microwell
1
β-Galactosidase Assay in Microbial Strains
2
Cellular Uptake and Cytotoxicity of AuNR-PEG
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The 786-0 (human renal
adenocarcinoma), A549 (human lung carcinoma), HepG2 (human liver carcinoma),
Hek293 (human nontumorigenic embryo kidney), NL20 (immortalised human
nontumorigenic lung), and THLE-3 (immortalised human nontumorigenic
liver) cell lines were obtained from ATCC and cultured as specified
by manufacturer’s recommendations at 37 °C in a 5% CO2 humidified environment. Further information on cell lines
is described in theSI .
For uptake
quantification of AuNR-PEG, 5000 cells were seeded in 96-well plates
(polystyrene clear flat bottom, ThermoFisher Scientific) and allowed
to grow for 2 days. For cytotoxicity assays, 2 × 104 cells were seeded in 24-well plates (polystyrene clear flat bottom,
Nunc Microwell, ThermoFisher Scientific) and allowed to grow for 3
days. For photothermal irradiation, 2 × 105 cells
were seeded in 6-well plates (polystyrene clear flat bottom, Nunc
Microwell, ThermoFisher Scientific) and allowed to multiply until
each well contained 8 × 105 cells.
Prior to
incubation with AuNR-PEG, wells were washed with appropriate
culture medium without supplements and AuNR-PEG were added, diluted
in culture medium without FBS for cancer cell lines and with 5–10%
FBS for healthy cell lines, at the concentrations specified for each
experiment.
adenocarcinoma), A549 (human lung carcinoma), HepG2 (human liver carcinoma),
Hek293 (human nontumorigenic embryo kidney), NL20 (immortalised human
nontumorigenic lung), and THLE-3 (immortalised human nontumorigenic
liver) cell lines were obtained from ATCC and cultured as specified
by manufacturer’s recommendations at 37 °C in a 5% CO2 humidified environment. Further information on cell lines
is described in the
For uptake
quantification of AuNR-PEG, 5000 cells were seeded in 96-well plates
(polystyrene clear flat bottom, ThermoFisher Scientific) and allowed
to grow for 2 days. For cytotoxicity assays, 2 × 104 cells were seeded in 24-well plates (polystyrene clear flat bottom,
Nunc Microwell, ThermoFisher Scientific) and allowed to grow for 3
days. For photothermal irradiation, 2 × 105 cells
were seeded in 6-well plates (polystyrene clear flat bottom, Nunc
Microwell, ThermoFisher Scientific) and allowed to multiply until
each well contained 8 × 105 cells.
Prior to
incubation with AuNR-PEG, wells were washed with appropriate
culture medium without supplements and AuNR-PEG were added, diluted
in culture medium without FBS for cancer cell lines and with 5–10%
FBS for healthy cell lines, at the concentrations specified for each
experiment.
3
Quantifying Bacterial Biofilm Formation
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The biofilm of WT, Δhfq, and hfq complementing strains of P. ananatis was quantified as previously described by Santander and Biosca (2017) (link) with slight modifications. An aliquot of 160 μl broth culture diluted to an OD600nm of 0.5 in half-strength LB [0.5% (w/v) NaCl, 0.5% (w/v) tryptone, and 0.25% (w/v) yeast extract; pH 7.2] was made into each well of a polystyrene 96-well microplate (NuncTM MicroWellTM, Thermo Scientific, Waltham, MA, United States) and incubated for 24 h under static conditions. Eight replicates per P. ananatis strain were included in each experiment with sterile half-strength LB broth serving as a negative control. Thereafter, the inoculated 96-well plates were inverted to remove the excess LB broth, air-dried, and incubated at 60°C for 40 min to heat-fix the biofilms. The biofilms were stained with 1% crystal violet (220 μl) for 15 min before being rinsed with distilled water. After rinsing and invert-air-drying the microplate, 220 μl of ethanol:acetone in 8:2 ratio was added to the wells to solubilize the crystal violet dye for 20 min at room temperature. The solubilized biofilm was measured at OD600 using Safire Microplate Reader (Tecan, Research Triangle Park, NC, United States), and this assay was repeated three times.
4
Quantifying Total Phenolic Content
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The total phenolic content (TPC) in methanol extracts was estimated using Folin–Ciocalteu (FC) reagent according to [5 (link)]. Gallic acid standard solution (960 μg/mL) was serially diluted 1:2 up to 60 μg/mL to construct the calibration curve. The FC reagent was diluted 1:10 with deionized water, while sodium carbonate was prepared as a 1 M solution. Briefly, 10 μL of extracts were pipetted in triplicate in wells of a 96-well microtiter plate (NuncTM MicroWellTM, ThermoFisher ScientificTM,Waltham, MA, USA,). After adding 100 μL of FC reagent and 80 μL of sodium carbonate to each well, the plate was allowed to incubate at room temperature in the dark for 20 min and read at 750 nm on a spectrophotometer (SpectraMAX 340PC, Molecular Devices Corporation, San Jose, CA, USA). TPC was expressed as mg Gallic Acid Equivalents (GAE)/g. Results are the mean values (LSmean ± SEM) of three replicates.
5
Immunocytochemical Staining Protocol
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Cells were grown in 96-well plates (NuncTM MicroWellTM, 167008, Thermo Fisher Scientific), fixed with 4% PFA (w/v) for 20 min, and then permeabilized in Triton X-100 0.2% diluted in PBS for 10 min. Cells were then blocked with 1% bovine serum albumin (BSA) (w/v) for 25 min. Cells were incubated with the primary antibody diluted in a blocking solution for 1 h and washed thrice in PBS. Cells were incubated with the secondary antibody (Invitrogen, Alexa FluorTM) diluted in blocking solution for 30 min and after washing thrice on PBS, 1 μg ml−1 of DAPI was added for 12 min and washed with PBS thrice.
6
Folin-Ciocalteu Assay for Polyphenols
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The Folin-Ciocalteu assay was performed on a 2103 EnVision Multilabel Reader (PerkinElmer Life and Analytical Sciences, Shelton, CT, U.S.A) in 96-well plates (Nunc MicroWell, Thermo Fisher Scientific, Waltham, MA, U.S.A) and the absorbance was measured at 630 nm. The assay was performed according to Hong et al. (2020) [36 (link)]. Extracts of buds, bark, and wood (10 μL) were mixed with 25 μL of 1 M Folin-Ciocalteu reagent (1:1 Folin-Ciocalteu 2 M:MilliQ), 25 μL 20 % sodium bicarbonate and 150 μL MilliQ using a multichannel pipette. The plate was incubated for 30 min at room temperature before reading. Gallic acid has been used to prepare standard curves in the range of 31.25, 62.5, 125, 250, 500, 750, and 1000 μg/ml. Total polyphenols concentration was calculated according to the gallic acid standard curve and expressed as mg equivalents of gallic acid/g of dry matter.
7
BCA Protein Quantification Protocol
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Protein concentration was determined following the Pierce BCA Protein assay kit (Thermo Scientific) protocol in 96-well plates (Nunc MicroWell; Thermo Fisher).
8
Sinularin's Impact on Cell Viability
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The viability of SK-HEP-1, Huh-7, and clone 9 cells after treatments with sinularin at 0, 0.5, 1, 5, 10, 25, 50, and 100 μΜ for 24, 48, and 72 h were evaluated using the MTT assay in triplicate. In brief, the cells were plated in 96-well microplates (Nunc™ MicroWell™, Thermo Fisher Scientific, Waltham, MA, USA) at a density of 5 × 103 cells per well. Following overnight incubation, the cells were treated with the indicated concentrations of sinularin for 24, 48, and 72 h. Subsequently, 20 µL of 5 mg/mL MTT solutions were added to wells, followed by incubating at 37 °C for 3 h. The absorbance from the resulting reduced product of MTT by viable cells was recorded at 570 nm using a microplate reader (Dynatech Laboratories, Chantilly, VA, USA). The relative cell viability (expressed in %) was calculated as the optical density of sinularin-treated cells divided by the optical density of untreated control cells and multiplied by 100. The percentage of viable cells was expressed as the mean ± standard error (SE).
9
Monitoring Bioluminescence in Chlamydomonas
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The transformant colonies were inoculated into 100 μL of TAP medium and maintained in 96 well plates (Nunc MicroWell, Thermo Fisher Scientific) for 3 days at 24°C in LL conditions (30–40 μmol m-2 s-1). Following this, 5 μL of the culture was transferred into 100 μL of fresh TAP medium containing D-luciferin (final concentration, 100 μM) in 96 well white plates (Nunc F96 MicroWell, Thermo Fisher Scientific). The plates were maintained at 24°C in LL conditions (30–40 μmol m-2 s-1) for 1 day before bioluminescence monitoring. Bioluminescence was monitored using a custom-made automatic bioluminescence apparatus [53 (link), 54 (link)].
10
Antimicrobial Susceptibility Testing of S. maltophilia
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S. maltophilia D457’s MIC values for PEP and DL-GA-3P were obtained on Mueller–Hinton (MH) medium by double dilution after 48 h at 37 °C. S. maltophilia D457 cells were grown in LB medium at 37 °C, with different compounds used at serial concentrations. The compounds used were fosfomycin (64, 32, and 16 μg/mL), PEP (1900, 950, and 475 μg/mL), and DL-GA-3P (340, 170, and 85 μg/mL). The stock solutions of the different compounds were diluted in LB medium to obtain the required concentrations. Growth was measured with a Spark 10M plate reader (Tecan, Männedorf, Switzerland) at OD600 in flat-bottomed transparent 96-well plates (Nunc MicroWell; Thermo Fisher; Waltham, MA, USA). Then, 10 μL of cell culture was inoculated in 140 μL of medium in each well, to a final OD600 of 0.01. The plates were incubated at 37 °C with 10s of shaking every 10 min. In all cases, a non-inoculated well containing the corresponding medium was included as a test of medium sterility.
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