Spleens of male and female 9–11 week old (mean age: 70 days) C57BL/6J mice (MFD-Diagnostics GmbH) were excised and transferred to 5 mL ice-cold RPMI 1640 (Thermo Fisher Scientific, Langenselbold, Germany). Tissue was disintegrated over a nylon cell strainer (70 μm; BD Biosciences, Heidelberg, Germany). Cell suspensions were centrifuged at 500 g for 5 min at 4 °C and resuspended in 2 mL 0.83% NH4Cl for 2 min at room temperature. Red blood cell lysis was stopped by adding 10 mL cold RPMI 1640. Splenocytes were collected by centrifugation, washed in RPMI 1640, and resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fischer Scientific). A quantity of 6 × 106 cells was seeded on 12 well polystyrene plates (Greiner, Frickenhausen, Germany) in 1 mL culture medium and cultured at 37 °C and 5% CO2. Splenocytes were stimulated as indicated in the figure legends.
Polystyrene plates
Manufactured by Greiner
Sourced in Germany, United States
Polystyrene plates are a type of laboratory equipment used for various applications. They are flat, disposable dishes made of polystyrene plastic material. The primary function of these plates is to provide a clean and stable surface for culturing cells, performing assays, and conducting other laboratory experiments.
Automatically generated - may contain errors
Lab products found in correlation
Nylon cell strainer, by BD (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Polyjet reagent, by SignaGen (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
Peroxidase conjugated anti golden syrian hamster igg antibody, by Rockland Immunochemicals (1 mentions)
O phenylenediamine dihydrochloride, by Merck Group (1 mentions)
Sensititre system, by Thermo Fisher Scientific (1 mentions)
96 well plate reader, by Molecular Devices (1 mentions)
7 protocols using polystyrene plates
1
Isolation and Culture of Mouse Splenocytes
2
Isolation of Murine Splenocytes
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Spleens obtained from 8- to 12-week-old male C57Bl/6 mice (MFD-Diagnostics GmbH) were excised and transferred to 5 ml ice-cold RPMI 1640 medium without FCS. Tissue was destroyed over a nylon cell strainer (70 µm; BD Biosciences, Heidelberg, Germany). Cell suspensions were centrifuged at 500 g for 5 min at 4°C and resuspended in 2 ml 0.83% NH4Cl for 2 min at RT. Red blood cell lysis was stopped by adding 10 ml cold RPMI 1640 medium without FCS. Splenocytes were collected by centrifugation, washed once with RPMI and resuspended in RPMI 1640, supplemented with 10% heat-inactivated FCS and 100 U/ml penicillin, 100 µg/ml streptomycin. Cells were seeded on 24 well polystyrene plates (Greiner) with 0.5 ml media in a concentration 6 × 106 cells/ml.
3
MEHCV Enzyme-Linked Immunosorbent Assay
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The wells of polystyrene plates (Greiner Bio-One) were sensitized with 20 ng purified rMEHCV dissolved in 100 µL 0.1 M sodium carbonate-bicarbonate buffer (pH 9.6). After incubation at 4°C for 16 h the coated wells were washed with PBST (PBS supplemented with 0.2% Tween 20, pH 7.2) and blocked for 2 h at 37°C with PBS containing 5% (w/v) dried skim milk powder and washed again with PBST. Subsequently, 100 µL of a dilution (100 µL PBST, 5% (w/v) dried skim milk powder, and 5 µL serum) was placed into the wells resulting in a final dilution of approximately 1/20. After incubation for 1 h at 37°C, the wells were washed with PBST and 100 µL of peroxidase-labeled goat anti-human immunoglobulin G conjugate (Sigma-Aldrich) diluted at 1 : 25,000 in PBS containing 5% (w/v) dried skim milk powder was added following incubation for 1 h at 37°C. The wells were again washed with 200 µL OPD by incubating for 30 min at room temperature. The optical densities (OD) were read at 450 nm. The results from the in-house kit were compared to those obtained from the Hepanóstika HCV Ultra® (Beijing, China) commercial kit.
4
Quantifying Readthrough Efficiency in DMD
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5,000 Cells were plated in each well of a 96-well black flat clear bottom Polystyrene plates (Greiner), a day before transfection. For transfection of the 8- or 9-well replicates of each sample, a pool containing 0.5 μg of the reporter and 0.5 μg of the snoRNA was prepared with polyJet reagent (Signagen Laboratories), according to the manufacturer's manual. As a calibration curve, mixes of the DMD reporter that does not contain a PTC (DMD no stop reporter) with the DMD PTC reporter were prepared, such that the no stop reporter was 0%, 0.5%, 1%, 2%, 4% or 8%. The fluorescence was measured 24 h post-transfection, in PBS, using an Infinite M200 Pro plate reader (Tecan). In order to assess the levels of readthrough based on the calibration curve, the ratio of the two fluorophores measurements of the samples were overlaid on the plot by calculating the intersection point of the average of the 8 measurements of each sample with the calibration line.
5
Quantification of Antibody Isotypes in Vaccinated Hamsters
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The ELISA assay was performed using a commercially available kit, 20 days after second immunization on sera from the vaccinated hamsters to determine the total antibody, IgG1 and IgG2 a isotypes. Briefly, the recombinant proteins (10 μg/mL) were adsorbed in PBS 1× buffer into 96-well polystyrene plates (Greinerbio-one, Fricken-hausen, Germany) overnight at 4°C. ELISA plates were washed three times with PBST (PBS with 0.05% [v/v] Tween 20) and then blocked with PBS containing 4% skim milk for 1 h at 25°C. The plates were then washed three times using PBST, Hamster's sera were added into the wells at dilutions of 1:100 to 1:12800, and incubated for 1 h at 25°C. Following rinses, Peroxidase-conjugated anti-golden Syrian hamster IgG antibody (Rockland) was added at a 1:6,000 dilution and incubated for one hour at room temperature. The plates were washed five times with PBST, o-phenylenediamine dihydrochloride (Sigma-Aldrich) substrate solution was added and the reaction was stopped with 4N H2SO4. Finally, optical density (OD) of mixtures was read at 492 nm by ELISA Reader.
6
Antibiotic Susceptibility Testing Protocol
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The antibiotic susceptibility test was performed using the N225 CARD (bioMerieux, Marcy l’Etoile, France) of the VITEK2 system for the following antibiotics: amikacin, ampicillin/sulbactam, cefepime, cefotaxime, ceftazidime, ciprofloxacin, colistin, gentamicin, imipenem, meropenem, minocycline, piperacillin, ticarcillin/clavulanic acid, and trimethoprim/sulfamethoxazole. In addition, the MIC for colistin was determined using the GNX3F plate of the Sensititre system (Thermo Fisher Scientific, Waltham, MA, USA) via the commercial broth microdilution kit and the standard broth microdilution. The breakpoint of the Clinical Laboratory Standard Institute (CLSI) M100-ED29 guideline was used to determine colistin resistance and susceptibility; MIC ≥4 μg/mL was taken to indicate colistin resistance. Reference MICs for colistin were determined using manual broth microdilution (BMD), performed according to the CLSI M07-ED11 guideline. MIC panels were prepared with pure colistin sulfate powder (Sigma–Aldrich, St. Louis, MO, USA) with two-fold dilutions in the range of 0.125–64 μg/mL in cation-adjusted MH broth (Becton–Dickinson, Sparks, MD, USA) in polystyrene plates (Greiner, Frickenhausen, Germany). Results were read after incubation in an aerobic atmosphere at 35 ± 2°C for 20–24 h. The results were interpreted based on the MICs according to the M100-ED29 guidelines.
7
Kinetic Characterization of Enzyme Mutants
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Initial-rate kinetics measurements were carried out on a Hewlett-Packard A8452A diode array spectrophotometer according to the conditions specified in Tables 123. For initial-rate measurements involving two varied substrate concentrations and for inhibition studies, a 96-well-plate-reader (Molecular Devices, Inc., Menlo Park, CA, USA) fitted with polystyrene plates (Greiner) was used. In order to check for possible adsorption of the enzyme that could interfere with the assay, the plates were coated with buffer containing 1% (w/v) bovine serum albumin and 2 % (v/v) Triton X-100. However, the rates of the enzyme reactions were not detectably influenced by pre-coating of the plates. Curve fitting and regression analyses were performed using the program SIMFIT [26] .
CDNB Glutathione 83 ± 2 345 ± 9 4.9 ± 0.5 5.0 ± 0.5 Inhibition constants were determined from inhibition studies for all enzymatically active mutants. Three inhibition experiments were done using three different concentrations of GSH (0.2 mM, 0.5 mM, 1.0 mM) and a constant concentration of CDNB (1 mM). The assay was performed at 30 • C in 100 mM sodium phosphate at pH 6.5, containing 2% (v/v) ethanol. IC 50 values were calculated as the concentration of the inhibitor that gives 50% of the reaction rate of the uninhibited reaction. Standard deviations of the measured parameters were less than 10%.
CDNB Glutathione 83 ± 2 345 ± 9 4.9 ± 0.5 5.0 ± 0.5 Inhibition constants were determined from inhibition studies for all enzymatically active mutants. Three inhibition experiments were done using three different concentrations of GSH (0.2 mM, 0.5 mM, 1.0 mM) and a constant concentration of CDNB (1 mM). The assay was performed at 30 • C in 100 mM sodium phosphate at pH 6.5, containing 2% (v/v) ethanol. IC 50 values were calculated as the concentration of the inhibitor that gives 50% of the reaction rate of the uninhibited reaction. Standard deviations of the measured parameters were less than 10%.
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