Mice were treated with 2.5 mg/ml enrofloxacin in drinking water for 2 weeks, followed by 0.8 mg/ml of amoxicillin and 0.114 mg/ml clavulanic acid in drinking water for a minimum of 2 further weeks prior to infection. Treatment with amoxicillin and clavulanic acid was then continued throughout the remainder of the experiment. For co-housing experiments, antibiotic treatment was discontinued and mice co-housed with naive or day 21 Hpb-infected SPF mice for a period of 3 weeks prior to HDM exposure. The effectiveness of antibiotic treatment was routinely assessed by plating of cecal contents under standard aerobic and anaerobic conditions and by microscopic analysis of fecal smears stained with Sytox Green nucleic acid stain (Life Technologies) and Gram staining (Sigma-Aldrich). Fecal smears were additionally checked for the presence of fungi by calcofluor white staining (Sigma-Aldrich).
Calcofluor white staining
Manufactured by Merck Group
Sourced in United States
Calcofluor white staining is a laboratory technique used to detect and visualize the presence of certain types of carbohydrates, particularly cellulose and chitin, in biological samples. It is a fluorescent dye that binds to these polysaccharides, making them visible under ultraviolet (UV) or fluorescent microscopy. The core function of Calcofluor white staining is to provide a simple and effective method for the identification and analysis of these structural components in various organisms and materials.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Gram staining, by Merck Group (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Cytation 3, by Agilent Technologies (1 mentions)
Paraformaldehyde solution, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Flow count fluorosphere, by Beckman Coulter (1 mentions)
4 protocols using calcofluor white staining
1
Antibiotic Modulation of Gut Microbiome
2
Biofilm Carbohydrate and Protein Analysis
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Carbohydrate content of 48-hour-grown biofilms was analyzed by using calcofluor-white staining (Sigma-Aldrich, St. Louis, MO, USA) and Congo red and safranin staining based on previous work. The protein content was assessed by SYPRO® RUBY (Thermo Fisher Scientific, Waltham, MA, USA) [38 (link)]. Briefly, after 10 min of exposure to each test-solution, biofilms were washed twice with PBS and then 1% Calcofluor-white, 0.1% Safranin and SYPRO RUBY were added to each well. The plates were maintained in the dark for 10 min and after that, the wells were washed twice with ultra-pure sterile water. Fluorescence readings for Calcofluor-white and SYPRO® RUBY were performed on Cytation 3 (Biotek, Winooski, VT, USA) at 430 nm/510 nm and 465 nm/630 nm, respectively. For Congo red and safranin, readings were performed in a spectrophotometer at 490 nm and 630 nm, respectively. The experiment was performed at two separate moments, each with three technical replicates.
3
Fungal Origin Confirmation by Flow Cytometry
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To confirm the fungal origin of populations analyzed, fungal stock aliquots (200 μl containing 2.105 fungal forms/μl) were thawed and incubated with 50 μl of calcofluor White staining (1 g/l, Sigma-Aldrich) during 15 min at 4 °C. Then, 400 μl of 2% (wt/vol) paraformaldehyde solution (ThermoFisher) was added to fix samples for 20 min at 4 °C. After 10-min 21,000 g centrifugation, supernatant was removed and pellet resuspended in the initial volume. Then, 25 μl of fungal suspension were mixed with 25 μl of flow count fluorospheres (Beckman Coulter) in 500 μl of PBS. Acquisition of microbeads and fungi was carried out using FACSCanto II flow cytometer (BD) and FACS Diva (BD) software.
4
Biofilm Formation Assay for Vibrio anguillarum
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The biofilm formation assay was performed in 96-well polystyrene microtiter-plates, as previously described [30] (link) with some modifications. Overnight cultures in LB20 were diluted with fresh LB20 medium to OD550 = 0.1 and inoculated into a 96-well plate (200 µl per well). The plate was incubated at 28°C for 48 hours, after which wells were washed three times with 300 µl sterile physiological saline to remove all non-adherent bacteria. The remaining attached bacteria were fixed with 200 µl of 99% methanol per well for 2 hours, and the plate was emptied and left to air dry overnight. Then, the plate was stained for 20 min with 200 µl of 1% crystal violet per well. Excess stain was rinsed off by placing the plate under running tap water. After the plate was air dried, the dye bound to the adherent cells was resolubilised with 200 µl of 95% ethanol per well. The absorbance of each well was measured at 570 nm. For the quantification of exopolysaccharides, a Calcofluor white staining (Sigma-Aldrich) was used as previously described [30] (link). For each assay, a minimum of three different V. anguillarum cultures were used for each treatment. The reported data are representative of three independent experiments.
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