To assess the relative survival of cells, glass beads recovered from stress treatment experiments were transferred to wells of a 24-well microtiter plate (Greiner Bio-One), with each well containing 350 μl of fresh medium and one added bead. The plates were incubated for 5 h at 24°C and 120 revolutions min−1 to allow bead-adhered cells to divide into the medium. An aliquot (300 μl) of the medium, now containing cells, was transferred to a 48-well microtiter plate, and further growth was monitored during incubation at 24°C with shaking in a BioTek Powerwave XS microplate spectrophotometer and OD600 measurement every 30 min. Resulting growth curves were used to infer back to a theoretical starting OD using multiple regression (22 (link)).
24 well microtiter plate
Manufactured by Greiner
Sourced in Germany
24-well microtiter plates are a type of laboratory equipment used for various cell-based assays and experiments. They provide a standardized format with 24 individual wells, allowing for multiple samples or conditions to be tested simultaneously. The plates are designed to be compatible with common laboratory equipment and procedures.
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Lab products found in correlation
Powerwave xs microplate spectrophotometer, by Agilent Technologies (1 mentions)
Cl 1000 ultraviolet crosslinker, by Analytik Jena (1 mentions)
Rnase h, by Thermo Fisher Scientific (1 mentions)
4 thio utp, by TriLink (1 mentions)
Facscalibur, by BD (1 mentions)
Mouse duoset elisa kit, by R&D Systems (1 mentions)
5 protocols using 24 well microtiter plate
1
Assessing Relative Cell Survival
2
Photo-crosslinking of Transcribed Plasmid
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Photo-crosslinking of transcribed plasmid was carried out as described (14 (link)) with modifications. Briefly, after transcription with 4-thio-UTP (TriLink BioTechnologies), samples were treated with or without 0.2-U/μl RNase H (Fermentas, Thermo Scientific) at 37°C for 15 min. They were then transferred to a 24-well microtiter plate (Greiner Bio-One, Germany), placed on ice in a UVP CL-1000 Ultraviolet Cross-linker (UVP), and irradiated for 20 min with 365-nm ultraviolet (UV) light at a distance of 4–5 cm. DNA was recovered with phenol/chloroform extraction and ethanol precipitation. Next, primer extension was performed with a 5′-FAM-ATTACAGGCGAACATACTTAC-3′ primer and Thermo Sequenase from the Cy5 Dye Terminator Cycle Sequencing Kit (GE Healthcare). Guanine and thymine ladders were prepared by primer extension in the presence of ddCTP or ddATP, in a 1/100 molar ratio to dCTP or dATP, respectively.
3
Murine Cytokine Response to Viral Antigens
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To assess the pattern of cytokines induced by antigens put into separate groups of mice, two weeks following the second injection, they were sacrificed under sterile conditions, and their spleen was extracted and homogenized. Shortly, (∼2 × 106) spleen cells have cultured in 24-well microtiter plates (Greiner, Germany) with or without 10 μg/ml of filtered FNOM and S proteins. After 72 h of incubation, the supernatants were gathered and kept at −70 °C till the cytokines were tested. The supernatants' concentrations of IL-17F, IL-22, IL-6, IFN-γ, TNF-α, IL-17A, and IL-10 were measured by a mouse 7 Plex cytokine assay kit (BioLegend, USA) using a flow cytometer (BD FACSCaliburTM, USA). The cytokine pattern was compared between different groups of mice.
4
Evaluation of Cytokine Responses and Bacterial Loads in Vaccinated Mice
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Six mice from each group were sacrificed two weeks after the last vaccination to evaluate the production of interferon-γ (IFN-γ), interleukin-4 (IL-4) and IL-17 cytokines using monoclonal antibodies against the cytokines in the supernatant of cultured splenocytes. In Summary, the spleen cells (3 × 105 cell/well) were cultured in 24-well microtiter plates (Greiner, Germany) under sterile conditions. Then 10 μg/mL filtered proteins were incubated for 72 h to collect the supernatants. Finally, the cytokine levels in the supernatants were measured using Mouse DuoSet ELISA kit according to the manufacturer’s instructions (R&D Systems).
The protection efficacy of the induced immune responses was examined in the vaccinated groups. In this UTI model, 6 mice in each vaccinated group were anesthetized and their bladders were emptied with gentle pressure and transurethrally challenged with 1 × 108 cfu/mL of bacteria using sterile polyethylene catheter. One week after inoculation, the mice were sacrificed and their homogenized bladders and kidneys were cultured in different dilutions to determine their bacterial loads.
The protection efficacy of the induced immune responses was examined in the vaccinated groups. In this UTI model, 6 mice in each vaccinated group were anesthetized and their bladders were emptied with gentle pressure and transurethrally challenged with 1 × 108 cfu/mL of bacteria using sterile polyethylene catheter. One week after inoculation, the mice were sacrificed and their homogenized bladders and kidneys were cultured in different dilutions to determine their bacterial loads.
5
Antimicrobial Barrier Assay for Food Films
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In order to evaluate the film's performance in preventing microbial superficial contamination of foods, a microplate antimicrobial barrier assay was used according to the methodology proposed by Pérez, Soazo, Balagué, Rubiolo, and Verdini (2014) (link). Five hundred microliters (500 μL) of trypticase soy agar (TSA, pH 5.5) with the addition of 50 mg/mL of 2,3,5-triphenyl tetrazolium chloride (TTC; Merck, Darmstadt, Germany) was poured into 24-well microtiter plates (Greiner Bio-One, Frickenhausen, Germany). The acidic condition of the media (pH 5.5) was selected to provide a pH value similar to those reported for several foods, like cheeses and meats (Han & Floros, 1997) (link). Bacterial suspension (∼5 × 10 3 CFU/mL) of Escherichia coli ATCC 25922, Salmonella Typhimurium ATCC 14028 or Listeria monocytogenes ATCC 19115 were prepared in saline solution (NaCl 0.85% wt/v) from overnight cultures of each bacterial strain and were then used for inoculation for the microbial barrier test 16 mm diameter PVC-based films discs were cut from and applied to the surface of the wells filled with the agar medium. Then, 10 μL of each bacterial inoculum were seeded on the film discs. Microplates were incubated at 37 °C for 24 h until red colored colonies were observed by the naked eye. Experiments were carried out in triplicate.
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