The biofilm formation ability assay was performed according to the methods by O’Toole with some minor modifications (Niemirowicz et al., 2016 (link)). An overnight culture of each isolate was incubated at 37°C/180 rpm up to the logarithmic phase with an OD600 value of 0.6, and the turbidity was adjusted to 0.5 McFarland standard, further diluted to 1:100, and CDB at 0, 32, 64, 128, 256, and 512 μg/mL concentrations were added, respectively. Then, 100 μL of the dilution was added to the 96-well polystyrene micro-test plate (Flat bottom with lid, Sterile; Corning, United States), and 3 replicate wells were set up. Following 24-h incubation at 37°C with shaking at 75 rpm, the upper planktonic bacteria was decanted, and biofilms attached to the well surfaces were stained with 100 μL of 1% (w/v) crystal violet solution (lot number: NO.20190324, Beijing Solarbio Biotechnology Co., Ltd., China) for 15 min. The bound dye was solubilized for 30 min with 100 μL of the eluent (95% absolute ethanol and 5% glacial acetic acid) and subsequently quantified by measuring the OD595 value by the Multiskan FC Microplate Reader.
Multiskan fc microplate reader
Manufactured by MultiSciences Biotech
The Multiskan FC Microplate Reader is a versatile instrument designed for absorbance measurements in microplates. It can perform a range of photometric analyses, including endpoint, kinetic, and spectral scanning, across multiple wavelengths. The Multiskan FC Microplate Reader is a reliable tool for various applications in life science research and clinical diagnostics.
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Lab products found in correlation
4 protocols using multiskan fc microplate reader
1
Biofilm Formation Ability Assay
2
Antimicrobial Susceptibility Profiling
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Susceptibility to biocides and antibiotics was evaluated by determination of minimum inhibitory concentrations (MICs) using a 96-well plate (Corning Incorporated) broth microdilution method developed by the UK Health Security Agency (UKHSA) (Bock et al., 2018 (link)). Doubling dilutions of antimicrobials were added to wells before inoculation with ca. 105 CFU mL−1 of overnight culture. Plates were incubated statically for 20 h at 37°C before OD600 nm was measured with a plate reader (Multiskan™ FC Microplate Reader). For each compound assayed, the MIC was defined as the lowest concentration which prevented measurable growth.
3
Treg Cell Suppression Assay
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A total of 1x105 of the sorted CD4+ T cells and CD4+ CD25+ Tregs were cultured and resuspended in 100 µl culture medium (RPMI 1640 supplemented with 10% FCS, 1% L-glutamine, HEPES 1 M and 1% penicillin-streptomycin) in sterile plastic 96-well, flat-bottom microwell culture plates (Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2. For proliferation analysis, CD4+ CD25+ Tregs and CD4+ T cells were cultured separately, and 100 µl control medium with or without phytohaemagglutinin (PHA) (cat. no. P8090; Beijing Solarbio Science & Technology Co., Ltd.) was added to the appropriate wells. The final concentration of PHA was 20 µg/ml. For suppression analysis, CD4+ T cells and CD4+ CD25+ Tregs were co-cultured at a 1:1 ratio (0.5x105 + 0.5x105) for 96 h in the same conditions. Tregs inhibitory capacity was assessed using CCK-8 assay (Beyotime Institute of Biotechnology). A 10 µl solution of CCK-8 reagent was added to the cells and incubated with 5% CO2 at 37˚C for 2 h. The absorbance of samples was then measured at 450 nm using a Multiskan™ FC microplate reader.
4
Chondrocyte Extracellular Matrix Evaluation
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After a 12-day micromass culture, the medium was changed, and the chondrocytes were
treated with 0, 10, and 40 μg/mL PPS for 72 hr. Alcian blue staining was performed to
evaluate ECM formation [8 (link)]. Briefly, the cells were
gently washed with PBS and fixed with 4% formaldehyde solution (Wako) for 30 min. After
rinsing with PBS, the micromasses were incubated with 1% Alcian blue solution
(Sigma-Aldrich) in 0.1 N hydrogen chloride (HCl; Wako) for 30 min, then washed thrice with
0.1 N HCl and once with distilled water. The color was inspected visually and
microscopically. Proteoglycan deposition was semi-quantified according to a technique
described by Griffin et al. [24 (link)].
The dye was solubilized in 6 M guanidine hydrochloride (Sigma-Aldrich) overnight at room
temperature and the absorbance was measured using a Multiskan FC microplate reader at 595
nm.
treated with 0, 10, and 40 μg/mL PPS for 72 hr. Alcian blue staining was performed to
evaluate ECM formation [8 (link)]. Briefly, the cells were
gently washed with PBS and fixed with 4% formaldehyde solution (Wako) for 30 min. After
rinsing with PBS, the micromasses were incubated with 1% Alcian blue solution
(Sigma-Aldrich) in 0.1 N hydrogen chloride (HCl; Wako) for 30 min, then washed thrice with
0.1 N HCl and once with distilled water. The color was inspected visually and
microscopically. Proteoglycan deposition was semi-quantified according to a technique
described by Griffin et al. [24 (link)].
The dye was solubilized in 6 M guanidine hydrochloride (Sigma-Aldrich) overnight at room
temperature and the absorbance was measured using a Multiskan FC microplate reader at 595
nm.
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