The protein concentration of cell lysates was determined using the bicinchoninic acid (BCA) assay. Protein standards (0.08, 0.1, 0.2, 0.4, 1 and 2 mg/ml) were prepared in 1% SDS lysis buffer using a 2 mg/ ml bovine serum albumin standard stock solution. 10 μl of blank (buffer alone), protein standards and samples were added to a Greiner Bio-One 96-well plate. 1:2 dilutions of protein samples were also included. 200 μl of developing solution (50:1 bicinchoninic acid:copper sulphate solution) was added to each well and incubated at 37° C for 30-60 min. Absorbance was measured using a microplate reader (Molecular Devices) and sample concentrations were determined from the standard curve.
Bio one 96 well plate
Manufactured by Greiner
Sourced in Austria
The Bio-One 96-well plate is a laboratory equipment designed for a variety of cell culture and assay applications. It provides a standardized format with 96 individual wells, allowing for multiple samples or replicates to be processed simultaneously.
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4 protocols using bio one 96 well plate
1
Protein Quantification via BCA Assay
2
Fluorescence Imaging of Cellular Changes
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To obtain fluorescence images using Hoechst 33342, JC-1, MDC, and AO, cells (1 × 10⁴) were seeded in a Greiner bio-one 96-well plate (Greiner, Kremsmünster, Austria) and cultured for 24 h until the cells adhered to the well and stabilized. Then, chrysophanol was added to the medium according to the determined concentration, applied to the cells, and cultured for 24 h. Hoechst 33342 was used to observe nuclear morphological changes. Cells were fixed with 4% paraformaldehyde 10 min, and 1 μg/mL of a Hoechst 33342 solution stained the nuclei of the cells at 37 °C. JC-1 was used for verifying the change of mitochondria membrane potential (ΔΨm). The JC-1 dye (2 μg/mL) stained for 30 min at 37 °C. To identify acidic vacuoles, an acridine orange (AO) agent (1 μg/mL) was used to stain the acidic vesicular organelles for 5 min. For autophagic fluorescence observation, the final concentration of 50 μM of the monodansylcadaverine (MDC) solution was stained with autophagic vacuoles for 30 min. After staining, cells were washed 3 times with PBS and mounted using glycerol. Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA) was used to observe changes in cellular fluorescence images. Cells were observed and photographed at ×200 magnification.
3
Permeability Assay for Dansyl Polymyxin and AR-12
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A. baumannii strains and K. pneumoniae strains were grown by inoculating colonies from freshly streaked Mueller-Hinton II agar plates into CAMHB at 37°C with shaking for ~16 hrs. A 1:100 dilution was subcultured in CAMHB and grown for additional 2–3 hrs to get log phage cultures. OD 600 was measured and volume of culture amounting to 0.5 OD in 1 ml was centrifuged and re-suspended in 1 mL of 5 mM HEPES buffer and kept at room temperature. Greiner Bio-One 96 well plates (catalog #655096, black with clear bottom) were used for testing the permeability of Dansyl polymyxin in the presence and absence of AR-12. A mix consisting of 5 mM HEPES buffer- pH 7·2, dansyl polymyxin (0.6 μM) and appropriate AR-12 concentration (1.25 × stock) was prepared. 80 μl of this mix was suspended in the wells in triplicates, following which 20 μl aliquots of the bacterial suspension in 5 mM HEPES buffer was added. Fluorescence was read at room temperature in a Tecan Sapphire II multimode plate reader, with filters of 320 nm (bandwidth 10 nm) for excitation and 500 nm (bandwidth 10 nm) for emission. To determine the fluorescence due to dansyl polymyxin uptake, the fluorescence from the wells containing the dansyl polymyxin and AR-12 but with no cells was subtracted from the wells containing the cells and the mix. The experiments were repeated two or more independent times to verify results.
4
Bacterial Outer Membrane Permeability
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Bacterial suspensions were prepared as described for the dansyl polymyxin permeability assay. Greiner Bio-One 96 well plates (catalog #655096, black with clear bottom) were used for testing the permeability of NPN in the presence and absence of AR-12. Stock concentration of NPN (5 mM) was made in acetone, following which a stock concentration of NPN (40 μM) in 5 mM HEPES-pH 7·2 was made from the 5 mM NPN stock. The 5 mM NPN stock can be stored in the −20°C for up to a month. A mix consisting of 5 mM HEPES buffer, NPN (40 μM) and AR-12 (10 × stock) was prepared for each concentration of AR-12 that was tested. 80 μl of this mix was suspended in the wells in triplicates, following which 20 μl aliquots of the bacterial suspension in 5 mM HEPES buffer was added. Fluorescence was read at room temperature in a Tecan Sapphire II multimode plate reader with filters of 355 nm (bandwidth 10 nm) for excitation and 405 nm (bandwidth 10 nm) for emission. To determine the fluorescence due to NPN uptake, the fluorescence from the wells containing the NPN and AR-12 but with no cells was subtracted from the wells containing the cells and the mix. The experiments were repeated two or more independent times to verify results.
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