To investigate cell growth and biofilm formation, 200 μl of culture broth containing serial-diluted oxacillin were prepared in a 96-well plate. Pre-cultured cells were inoculated (1% v/v) and the plate was incubated at 37°C for 24 h without shaking. For cell growth, optical density was measured by using a 96-well plate reader (Thermo Fisher Scientific, USA). Biofilm formation was analyzed by using crystal violet [25 (link)]. After the supernatant was gently removed, biofilm fixation was carried out with methanol and subsequently air-dried. Thereafter, 0.2% of crystal violet solution was added to each well to stain the biofilm. After 5 min, the remaining dye was removed with distilled water and the absorbance was measured at 595 nm using a 96-well microplate reader (Thermo Fisher Scientific) [24 (link)]. For disc diffusion method, to inoculate each strain onto TSB agar plate sterile swab was used. Using sterile forceps, discs with 30 μg of oxacillin were carefully distributed on the inoculated plates. The plates were kept on the clean bench for 30 min to allow pre-diffusion of the antibiotics, then incubated aerobically at 37oC for overnight. The zones of inhibition were measured using a meter rule and compared with CLSI guidelines [26 (link)].
96 well microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 96-well microplate reader is a laboratory instrument used to measure the absorbance or fluorescence of samples in a 96-well microplate format. It is designed to quantify the optical properties of solutions, such as the concentration of a particular substance or the activity of an enzyme. The microplate reader can be used for a variety of applications, including enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and other colorimetric or fluorometric analyses.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate reader, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides lps from e coli, by Merck Group (1 mentions)
Elisa kit for tnf α, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
Atcc tib 71, by American Type Culture Collection (1 mentions)
Il 1β, by BD (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Fluorescein isothiocyanate labeled dextran, by Merck Group (1 mentions)
Fluorescence spectrophotometer, by Shimadzu (1 mentions)
7 protocols using 96 well microplate reader
1
Evaluating Antimicrobial Resistance and Biofilm Formation
2
Cell Viability Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, cells were seeded at 5000 cells/well into a 96-well plate and incubated overnight. After exposure to different treatments, MTT solution (final concentration 0.5 mg/ml) was added to each well, and the samples were incubated for another 4 h. Subsequently, the supernatant was removed and cells were dissolved in 150 μl dimethyl sulfoxide (DMSO). Finally, absorbance at 570 nm was measured by using a 96-well microplate reader (Thermo Scientific).
3
Antimicrobial Screening of Bacterial Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells were purchased from the American Type Culture Collection (ATCC TIB-71). Live bacterial cultures of Corynebacterium xerosis, Enterococcus faecalis, Bacillus subtilis, Staphylococcus epidermidis, Bacillus megaterium, Staphylococcus aureus, Escherichia coli and Salmonella typhimurium were purchased from Ward’s Science (Rochester, NY, USA). An additional E. coli ΔtolC mutant was also used is this study; the mutation to the TolC multi-drug efflux pump decreases its activity and inhibitory activity against this strain provides evidence whether or not drug-efflux activity possessed by Gram-negative bacteria reduces the inhibitory activity of the extract [16 (link)]. Lipopolysaccharides (LPS) from E. coli were purchased from Sigma-Aldrich. A tetrazolium dye (WST-8) cell viability assay kit (CCK-8) was purchased from Dojindo Laboratories and an ELISA kit for TNF-α was purchased from eBioscience. All measurements from proliferation assays and ELISA experiments were performed using a 96-well microplate reader (Thermo-Fisher, Inc., Waltham, MA, USA). Two-way analysis of variance was performed using four replicate measurements of cell proliferation for each control and treatment group and three replicate ELISA measurements for each control and treatment group.
4
Cytokine Detection in Cell Medium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell medium was collected into tubes and stored at -20°C until further usage or detection. Released cytokines IL-6, IL-7, IL-8, TNF-α, ICAM-1, and VEGF in cell medium were detected using human cytokine ELISA detection kits (Neobioscience, China). When detected, 20 μL cell medium was used to determine the content of human cytokines according to the manufacturer's instructions. Fluorescent intensity was measured by using a 96-well microplate reader (Thermo Fisher, USA).
5
Cytokine Profiling of Cell-Derived Secretome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell-free supernatants derived from the coculture of RAW264.7 and 4T1 cells were stored at −70°C prior to measurement of cytokine production. The amounts of IL-6 (Invitrogen, Carlsbad, California), IL-1β (BD Science, San Diego, California), and tumor necrosis factor α (TNF-α; R&D System, Minneapolis, Minnesota) produced in the CM were determined with commercial enzyme-linked immunoassay kits according to the manufacturer’s protocol. The absorbance at 450 nm was obtained using a 96-well microplate reader (Thermo Scientific, Rockford, Illinois).
6
Endotoxin and Intestinal Permeability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum from the portal vein was analyzed for endotoxin using the endpoint assay method (LDL QCL 1000; BioWhittaker, Walkersville, MD). Samples were eluted for 1 hour in 30 mL pyrogen‐free PBS at room temperature. Dilutions were assayed using a 96‐well microplate reader (Thermo Fisher Scientific Inc., Minneapolis, MN).
To measure intestinal permeability, 125 mg/kg body weight of 40 kDa fluorescein isothiocyanate ‐labeled dextran (Sigma‐Aldrich, Oakville, Canada) was administered by oral gavage 4 hours before being killed. Blood was collected by portal venipuncture. Plasma fluorescence was determined with a fluorescence spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) at 490‐nm excitation wavelength and 520‐nm emission wavelength using a series of known fluorescein isothiocyanate ‐dextran concentrations diluted in rat plasma.
To measure intestinal permeability, 125 mg/kg body weight of 40 kDa fluorescein isothiocyanate ‐labeled dextran (Sigma‐Aldrich, Oakville, Canada) was administered by oral gavage 4 hours before being killed. Blood was collected by portal venipuncture. Plasma fluorescence was determined with a fluorescence spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) at 490‐nm excitation wavelength and 520‐nm emission wavelength using a series of known fluorescein isothiocyanate ‐dextran concentrations diluted in rat plasma.
7
Antimicrobial Susceptibility and Biofilm Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate the antimicrobial susceptibility and biofilm formation, 200 μl of culture broth containing serially diluted oxacillin was prepared in a 96-well plate. Precultured cells were inoculated (1% v/v), and the plate was incubated at 37°C for 24 h without shaking. Cell optical density was measured using a 96-well plate reader (Thermo Fisher Scientific, USA). Biofilm formation was analyzed using crystal violet staining [29 (link)]. After the supernatant was carefully removed, biofilm fixation was performed with methanol and subsequently air-dried for 24 h. Thereafter, a 0.2% crystal violet solution was added to each well to stain the biofilm for 5 min. The remaining dye was removed and washed twice with distilled water. Finally, absorbance was measured at 595 nm using a 96-well microplate reader (Thermo Fisher Scientific) [28 (link)]. As chlorothymol is insoluble in common solvents, including water, ethanol was used as the solvent. Due to the potential toxic effect of ethanol on the cells, the same amount of ethanol without the compound was added to the control, and an inhibitory effect test was performed [30 (link)]. Ethanol did not show any inhibitory effect on cell growth even at the volume used with a compound concentration of 512 μg/ml (Fig. S1 ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!