DNA from bacterial isolates was extracted using the boiling method (Abrahamian et al., 2018 (link)) modified by the addition of a chloroform extraction. Single colony glycerol stocks were streaked and grown on nutrient broth yeast (NBY) medium for 48–72 h. Bacteria from a single colony was transferred into a 1.5 ml microcentrifuge tube and re-suspended in 400 ml of sterile water. Tubes were boiled at 100°C for 15 min in a dry-bath (Thermo Fisher Scientific, Waltham, MA, United States), and then immediately placed on ice for 5 min followed by centrifugation at 10,000 rpm for 5 min. An aliquot of 100 μL of isolated DNA was mixed with 300 μL of Tris EDTA buffer [10 mM Tris, 0.1 mM EDTA], and 400 μL of chloroform isoamyl alcohol (24:1) followed by centrifugation at 5,000 × g for 15 min. About 150 μL of the aqueous phase was transferred to a 96-well round bottom microtiter plate (Corning Inc., Kennebunk, ME, United States) containing 15 μL of sodium acetate (3M, at pH 5.3) and 330 μL of 70% ethanol followed by centrifugation at 5,000 × g for 10 min for DNA precipitation. Plates were air-dried and 100 μL of ddH20 was added. For DNA isolation of plant material we used methods described in Bernal et al. (2020) (link).
96 well round bottom microtiter plate
Manufactured by Corning
Sourced in United States
96-well round-bottom microtiter plates are a type of laboratory equipment used in various scientific experiments and assays. These plates feature a circular well design with a rounded bottom, providing a consistent surface area for sample containment and accurate volume measurements. The 96-well format allows for the simultaneous processing of multiple samples, improving efficiency and throughput in research and analytical settings.
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Lab products found in correlation
Dhr 123, by MoBiTec (5 mentions)
Accurie c6 flow cytometer, by BD (2 mentions)
Dry bath, by Thermo Fisher Scientific (1 mentions)
Parcan, by Septodont (1 mentions)
Rpmi media, by Merck Group (1 mentions)
Pkh26 dye, by Merck Group (1 mentions)
Modfit software, by Verity Software House (1 mentions)
Facscalibur, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
11 protocols using 96 well round bottom microtiter plate
1
Bacterial DNA Extraction with Chloroform
2
Screening Prestwick Library for Antibacterial Hits
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The Prestwick chemical library was screened to identify hits active against clinically relevant human bacterial pathogens as specified above. The compounds (2 μL) were added to 96-well round-bottom microtiter plate (Corning) followed by 98 μL of bacterial inoculum, resulting in a final drug concentration of 50 μM in the primary screen. Wells containing only cells (positive control) or medium were included on each assay plate, and the plate was incubated at 37°C for 18 to 24 h to determine MIC. After the initial hit determination, the hit compounds were serially diluted to determine the MIC of specific compounds.
3
Camel Granulocyte ROS Production Assay
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The production of ROS metabolites was measured in 96-well round-bottom microtiter plates (Corning, NY, USA) as previously described [29 (link)]. Camel granulocytes (1 × 106/100 μL/well) were incubated in 50 μL RPMI culture medium alone or in a medium containing T. evansi (4 × 106 parasite/mL) for 30 min (37 °C, 5% CO2). After 15 min of incubation, dihydrorhodamine (DHR) 123 (Mobitec, Goettingen, Germany) was added to the cells at a final concentration of 750 ng/mL. The cells were washed in RPMI medium, and ROS production was analyzed by flow cytometry.
4
Antifungal Susceptibility of NaOCl and EDTA
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MIC was determined using a broth microdilution method according to the M27-A3 standard for fungi [32 ] (CLSI). Briefly, cells were adjusted to the desired density of 2 × 104 cells/mL in Roswell Park Memorial Institute (RPMI) media (Sigma–Aldrich, Dorset, UK). A series of two-fold dilutions of 3% (30,000 ppm) NaOCl (Parcan; Septodont, Saint-Maur-des-Fosses, France) and 17% (EDTA [ENDO-SOLution, Stalowa Wola, Poland]) were performed using 96-well round-bottom microtiter plates (Corning Incorporated, Corning, NY, USA). The plates were at 37 °C. After 24 h, the MIC concentration was determined as the lowest concentration that prevents visible growth.
5
Antigen-specific T Cell Proliferation Assay
6
Quantifying Leukocyte ROS Generation
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ROS generation was performed in 96-well round-bottom microtiter plates (Corning, NY, USA) [32 (link)]. Stimulated and non-stimulated leukocytes (1 × 106/well) were incubated for 20 min (37 °C, 5 % CO2) with the ROS-sensitive dye dihydrorhodamine (DHR)-123, (750 ng/ml final, Mobitec, Goettingen, Germany). After incubation, cells were washed with MIF buffer, and the percentage of ROS-positive cells and the relative amount of generated ROS was determined by flow cytometry (Accurie C6 flow cytometer, BD Biosciences) after acquisition of 100 000 events (Fig. 2 A-B).
7
Camel Leukocyte ROS Generation
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ROS generation was performed in 96-well round-bottom microtiter plates (Corning, NY, USA) as described earlier [28 (link)] with modifications. Separated camel leukocytes (1 × 106 / well) in RPMI medium were incubated with heat killed Staphylococcus aureus (50 bacteria/cell) for 20 min (37 °C, 5% CO2). For the detection of ROS, dihydrorhodamine (DHR) 123 (Mobitec, Goettingen, Germany) was added to the cells (150 ng / ml final). To identify monocyte subsets, cells were labeled with monoclonal antibodies to CD14 and MHCII (see above). After washing, cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, California, USA). The relative amount of generated ROS was determined by the mean green fluorescence intensity of gated monocyte subsets (based on CD14 and MHCII expression) after acquisition of 100,000 events (n = 15 animals).
8
ROS Generation and Quantification in Leukocytes
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ROS generation was performed in 96-well, round-bottom microtiter plates (Corning, NY, USA) [10 (link)]. Leukocytes (1×106/well) were incubated for 30 min (37°C, 5% CO2) with heat-killed S. aureus bacteria (30 bacteria/cell) in the presence of ROS-sensitive dye dihydrorhodamine (DHR)-123 (750 ng/mL final; Mobitec, Goettingen, Germany). For monocyte identification, the cells were incubated with APC-conjugated monoclonal antibodies specific to CD14. After incubation, the cells were washed with PBS, and the percentage of ROS-positive cells and the relative amount of generated ROS were determined by flow cytometry (Accurie C6 Flow Cytometer; BD Biosciences, USA) after the acquisition of 100,000 events.
9
Leukocyte ROS Generation Assay
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ROS generation was measured in 96-well round-bottom microtiter plates (Corning, NY, USA) as previously described (37 (link)). Leukocytes separated from heat-stressed whole blood or heat-stressed leukocytes (1 × 106/well in RPMI medium) were incubated in duplicates for 20 min (37°C, 5% CO2) with heat-killed S. aureus (30 bacteria/cell) in the presence of the ROS-sensitive dye dihydrorhodamine (DHR)-123 (1 μg/mL final, Mobitec, Goettingen, Germany). After incubation, labeled cells were washed with PBS and the relative amount of generated ROS was determined by flow cytometry (Accuri C6 flow cytometer, BD Biosciences) after the acquisition of 100 000 events (gated leukocytes).
10
Antifungal Efficacy of Sulfur Nanoparticles
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Antifungal activity of the sulfur nanoparticles was determined using the broth microdilution method according to Clinical and Laboratory Standard Institute ( 2008) guidelines, with some modifications. Synthesized sulfur nanoparticles were diluted with 2% v/v xylene in distilled water to achieve a concentration of 200 mg/ml. Serial 2-fold dilution of sulfur nanoparticles was carried out with SDB in 96-well round-bottom microtiter plates (Corning Incorporated, USA) and 50 µl of fungal suspension was added to wells. The well which contains SDB and fungal suspension was considered as positive growth control, while the well containing only SDB was used as negative growth control. The final concentrations of sulfur nanoparticles were 50.000-0.098 mg/ml. The 2% v/v xylene in distilled water was used as diluent control. The plates were incubated at 30ºC for 72-96 h. The minimal inhibitory concentration (MIC) was considered as the lowest concentration of antifungal agents that inhibited visible growth of tested fungus. The mixture of no visible growth wells was transferred to SDA and incubated at 30ºC for 72-96 h. The minimum fungicidal concentration (MFC) was determined from the lowest concentration of antifungal agents that inhibited growth on SDA. Ketoconazole was used as antifungal controls. All tests were performed in triplicate.
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