Antimicrobial activity of BPI was determined in 3 to 5 independent experiments essentially as described before (Aichele et al., 2006 (link)). Briefly, S. Typhimurium and Pseudomonas aeruginosa were grown over night (O/N) in LB or TSB, respectively. After re-inoculating the strains 1:100 in fresh medium, cultures were allowed to grow to mid log phase (~2.5 h) and washed twice in PBS (PAA, Pasching, Austria). Assays were performed in 96-well microtiter plates (#655180, Greiner Bio-One, Frickenhausen, Germany) using 10,000 CFU/mL and the indicated amount of BPIF protein or buffer control in a total volume of 50 µL PBS per well. The plates were incubated for the indicated time points swimming in a water bath at 37°C. Thereafter, an aliquot of the bacteria was plated on LB-plates and enumerated the following day.
Phosphate buffered saline (pbs)
Manufactured by Greiner
Sourced in Germany
PBS (Phosphate-Buffered Saline) is a common laboratory buffer solution used to maintain the pH and osmolarity of biological samples. It is a mixture of sodium phosphate and sodium chloride in water, formulated to provide a physiologically compatible environment for cells and tissues.
Automatically generated - may contain errors
4 protocols using phosphate buffered saline (pbs)
1
Antimicrobial Activity Assay of BPI
2
Influenza Virus Antigen ELISA Assay
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We used ULTRIX with a purified influenza virus, as well as H1N1 and H3N2 antigens, to prepare for the vaccines. Antigens (1 μg/mL) were adsorbed in 96-well plates in PBS (Greiner Bio One GmbH, Frickenhausen, Germany) at 4 °C for 12 h. Then, they were washed in PBST and blocked by 1% casein solution in a wash buffer for 60 min at room temperature. After that, serum samples were added in a three-fold serial dilution starting at 1:30 and incubated for 60 min at 37 °C. After washing, we added rabbit antibodies against mice IgG conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 60 min at 37 °C. Then, we washed the plate and added a diluted TMB substrate (Amresco LLC, Solon, OH, USA). After terminating the reaction via a stop solution, we measured the optical density at a wavelength of 450 nm using a reader device for ELISA (ChroMate Awareness Technology Inc., Palm City, FL, USA). Graphs were constructed using GraphPad Prism 6.0 and Excel 2016.
3
Quantifying Apoptosis in PASMCS Cells
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Flow cytometry was used to detect and quantify the apoptotic rate of the PASMCS cells, by staining with Annexin V-FITC (Roche, Mannheim, Germany) and PI (propidium iodide) (Roche, Mannheim, Germany). After harvesting, the PASMCS cells (1×104) were washed with PBS (Greiner, Bahlingen, Germany) and incubated with Annexin V-PI (Roche, Mannheim, Germany) at room temperature for 15 min in the dark. A Becton Dickinson fluorescence-activated cell sorter (BD Biosciences, San Jose, CA) with 585/42 nm (PI) and 530/30 nm (FITC) emission filters was used to detect the PASMCS cells. Becton Dickinson Cell Quest software was used to analyze the percentage of apoptosis. Three independent experiments were performed.
4
ELISA for Eukaryotic Protein Antibodies
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The eukaryotic proteins RBD and S-trimer were used as antigens for ELISA, which were kindly provided by the laboratory of immunochemistry of the FBSI SSC VB Vector of Rospotrebnadzor (Russia). The RBD protein (1 μg/mL) and S-trimer (1 μg/mL) were adsorbed in the wells of a 96-well plate in PBS (Greiner Bio One GmbH, Frickenhausen, Germany) at 4 °C for 12 h, then washed with PBST and blocked with 1% casein solution in wash buffer for 60 min at room temperature. After that, serum samples were added in a threefold serial dilution, starting at 1:50, and incubated for 60 min at 37 °C. After washing, rabbit anti-mouse IgG antibodies conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated for 60 min at 37 °C. The plate was washed and a solution of TMB substrate (Amresco LLC, Solon, OH, USA) was added to it. After stopping the reaction with the stop solution, the optical density was measured at a wavelength of 450 nm using an ELISA reader (ChroMate Awareness technology Inc., Palm City, FL, USA). The graphs were built using GraphPad Prism 6.0 and Excel 2016.
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