MWCNT toxicity to P. aeruginosa was assessed by measuring membrane integrity using the LIVE/DEAD Bac Light Bacterial Viability Kit L7012, reductase activity using the BacLight™ RedoxSensor™ Green Vitality Kit (both from Molecular Probes, Invitrogen, CA, USA) and growth by measuring the time course optical density (600 nm). Viability of T. thermophila upon direct exposure to MWCNTs in acute conditions (non-growing culture) was assessed by cell counting and membrane integrity as in P. aeruginosa above. Experimental details are in the SI .
Baclight redoxsensor green vitality kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BacLight RedoxSensor Green Vitality Kit is a fluorescence-based assay designed to assess the respiratory activity of bacteria. The kit utilizes a redox-sensitive dye that measures the electron transport chain activity, providing an indication of cell viability and metabolic status.
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Lab products found in correlation
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Ti2 inverted fluorescence microscope, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Facscalibur, by BD (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Lsrfortessa, by Beckman Coulter (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
12 well flat bottomed polystyrene plates, by Corning (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Densimat apparatus, by bioMérieux (1 mentions)
11 protocols using baclight redoxsensor green vitality kit
1
MWCNT Toxicity to Bacteria and Protozoa
2
Determining Bacillus subtilis Redox Status
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RedoxSensor activities of B. subilis 3610 were determined using a BacLight RedoxSensor Green Vitality Kit (Molecular Probes, Eugene, OR, USA). Overnight cultures of B. subtilis 3610 were treated with the indicated concentrations of ZnO NPs for 3 h at 37°C. Bacterial cells were then washed and diluted 10-fold in 1X PBS buffer, then mixed with 1 μl of RedoxSensor Green reagent and vortexed. To assess membrane integrity, 1 μl propidium iodide was added, and the mixture was incubated in the dark at room temperature for 5 min. Stained cells (10 μl) were spotted onto a clean slide and covered with a poly-L-lysine treated coverslip. Slides were observed under a Leica TCS-SP2 laser-scanning confocal microscope at a magnification of 630X.
3
TEM and Viability Analysis of Bacterial Cells
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For TEM analysis, cells grown to OD600 = 2.0 were washed twice with PBS. The cells in the three treatment groups were collected and fixed overnight with 2.5% glutaraldehyde at 4 °C and then embedded in 2% agarose after centrifugation at 3000× g. Thin sections of the samples were stained with uranyl acetate for 15 min and observed using a Hitachi H-7650 transmission electron microscope (Hitach, Tokyo, Japan).
Cell viability after heat treatment was studied by staining with specific fluorochromes followed by epifluorescence microscopy. Cells were stained using a BacLightTM RedoxSensorTM Green Vitality Kit (ThermoFisher, MA, USA) containing PI and RSG. D. radiodurans cells were washed with PBS. This kit is convenient and easy-to-use for monitoring the viability of bacterial populations as a function of cell membrane integrity. Cells with a compromised membrane, which are considered dead or dying, stain red (PI), whereas cells with an intact membrane stain green (RSG). A fluorescence assay was performed using a 100× oil immersion lens with a Nikon Ti2 inverted fluorescence microscope and processed with the NIS-Elements (Nikon, Tokyo, Japan).
Cell viability after heat treatment was studied by staining with specific fluorochromes followed by epifluorescence microscopy. Cells were stained using a BacLightTM RedoxSensorTM Green Vitality Kit (ThermoFisher, MA, USA) containing PI and RSG. D. radiodurans cells were washed with PBS. This kit is convenient and easy-to-use for monitoring the viability of bacterial populations as a function of cell membrane integrity. Cells with a compromised membrane, which are considered dead or dying, stain red (PI), whereas cells with an intact membrane stain green (RSG). A fluorescence assay was performed using a 100× oil immersion lens with a Nikon Ti2 inverted fluorescence microscope and processed with the NIS-Elements (Nikon, Tokyo, Japan).
4
Cellular Redox Activity Profiling
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The cellular redox activity was studied after 4 h of antibiotic treatment at 5× MIC using RSG (Redox Sensor Green) and PI (Propidium iodide) staining. Briefly, the overnight grown culture was diluted to 0.1 OD at 600 nm in 3 ml LB and treated with antibiotic treatments as per the above-mentioned protocol. After 4 h of antibiotic treatment, 3 ml of sample was aliquoted, washed with PBS twice, and resuspended in 300 µl of PBS. For flow cytometry, the cells were stained using RSG (1 µl) and PI (1 µl of 1:100 diluted) dyes (BacLightTM RedoxSensorTM Green Vitality Kit Thermo Fisher Scientific Inc., Waltham, MA, USA) for 10 min in dark condition. No antibiotic treatment was used as a control. The flow cytometry (Becton Dickinson FACS Calibur, New Jersey, United States) was performed for the detection of fluorescence signals using FLI and FL3 channels. The RSG-positive cells were plotted in a histogram against the FL1 channel. The data were analysed using three independent biological triplicates27 (link).
5
Quantifying Microbial Metabolic Activity
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The analysis of metabolic activity was based on the procedure described by Cyplik et al. (2013) [42 (link)]. Approximately 1 mL of the culture medium was taken for analysis after 24 and 168 h of incubation. The samples were centrifuged (5 min, 3500 rpm) to separate the microbial cells. After decanting the supernatant, the pellet was washed twice using 1 mL of phosphate buffered saline (Sigma-Aldrich) and centrifuged (5 min, 3500 rpm). Then, the cells were suspended in 250 μL of phosphate buffered saline (Sigma-Aldrich) and 1 μL of Redox Sensor Green dye from the BacLight ™ RedoxSensor ™ Green Vitality Kit (Thermo Fisher Scientific, Waltham, MA, USA) was added to each sample. The systems were incubated for 10 min at room temperature. Fluorescence in the stained samples was measured using a BD FACS Aria ™ III flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The percentage ratio of active population (Q2) was determined by gating point plots of median values of green fluorescence signals (FITC-A) versus medians of light scattering values (SSC-A) based on a comparison to a previously prepared negative sample that contained metabolically thermally inactivated dead cells.
6
Evaluating Bacterial Redox Activity
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The reductase activities of S. aureus SA113 and E. coli K12 were determined using a BacLight™ RedoxSensor™ Green Vitality Kit (Thermo Fisher, Waltham, MA, USA) [43 (link),44 (link)]. Overnight cultures of approximately 1 × 107 CFU/mL in 25 mL of MHB were supplemented with the indicated concentrations of rGO for 3 h at 175 rpm and 37 °C. Cells were washed with 1X PBS buffer twice and diluted 10-fold with the same buffer. For E. coli K12, 1 µL of RedoxSensor™ Green reagent and 1 µL PI were added to the mixture. For S. aureus SA113, 1 µL of 10-fold diluted RedoxSensor™ Green reagent and PI dilution were added to the mixture. This was followed by incubation in the dark at room temperature for 5 min for the assessment of cell membrane integrity. Stained cells (1 mL) in PBS were assayed by flow cytometry using an Attune™ NxT Flow Cytometer (Thermo Fisher). Fluorescence filters and detectors were all standardized with green fluorescence collected in the FL1 channel (530 ± 15 nm) and red fluorescence collected in the FL3 channel (>650 nm). Data were analyzed using Attune™ NxT Flow Cytometer software.
7
Evaluating Bacterial Redox Activity
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Reductase activities of B. subtilis 3610 and B. thuringiensis 407 were determined using a BacLight RedoxSensor Green Vitality Kit (ThermoFisher, Waltham, MA, USA). Overnight cultures of approximately 1 × 107 CFU/mL in 50 mL MHB were supplemented with the indicated concentrations of ZVI NPs for 3 h at 175 rpm and 37 °C. Cells were washed with 1 × phosphate-buffered saline (PBS) twice, diluted 10-fold with the same buffer, and eventually mixed well with 1 μL of RedoxSensor Green reagent. Additionally, 1 μL of propidium iodide (PI) was added to the mixture, which was incubated in the dark at room temperature for 5 min before assessing cell membrane integrity. Stained cells (1 mL) in PBS were assayed with a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA). In addition, fluorescence filters and detectors were all standardized with green fluorescence collected in the FL1 channel (530 ± 15 nm) and red fluorescence collected in the FL3 channel (> 650 nm). Data were analyzed using FACSCalibur Flow Cytometer software. All parameters were collected as logarithmic signals. All measurements were performed in two separate experiments.
8
Assessing E. coli Metabolic Activity
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To ascertain metabolic activity, E. coli MG1655/pCV1-mqsRAC was grown to a turbidity of 0.5 at 600 nm, and T2 phage was added (MOI 0.1) for 1 h. Cells were washed, resuspended in PBS, stained for metabolic activity (BacLight RedoxSensor Green Vitality Kit, Thermo Fisher Scientific Inc., Waltham, MA, USA), which measures the cellular redox state. The fluorescence signal of 100,000 cells was analyzed by flow cytometry (Beckman Coulter LSRFortessa) using the FL1 (530 ± 30 nm).
9
Viability Assay for Borrelia burgdorferi
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B. burgdorferi strains were grown in BSK + RS at 35°C to ~7 × 107 cells ml-1 before collection at 8,000 x g for 10 min at 4°C. Cell pellets were resuspended in warm RPMI and incubated at 35°C for 16 h. Cultures were then stained with BacLight RedoxSensor Green and propidium iodide according to the manufacturer’s instructions (1 μl RedoxSensor Green and 1 μl propidium iodide in 1.0 ml culture) from the BacLight RedoxSensor Green Vitality kit (Invitrogen). Cells were stained for 10 min at 35°C, collected by centrifugation (13,000 x g, 5 min, 4°C) and cell pellets resuspended in 1.0 ml PBS. 10 μl of cells were wet-mounted on a slide and live cells imaged using an Olympus BX51 fluorescence microscope with 100x/1.30 NA objective. Images were processed using ImageJ (National Institutes of Health; http://rsbweb.nih.gov/ij/ ) and Pixelmator (Pixelmator Team, Ltd). The mean percent cells stained with RedoxSensor Green and mean percent round body (RB) cells were determined from three independent biological replicates where at least 100 cells were enumerated for each strain. Significant difference between the means was determined by one-way ANOVA with a Tukey’s post-hoc test.
10
Quantifying S. aureus Viability under Compound Treatment
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An overnight culture of S. aureus SH1000 was diluted 500-fold into BHIG broth 5% DMSO, with or without test compounds (20 μM JBD1, 20 μM ANG1, 20 μM JBD1 and 100 μM MK-7, or 100 μM MK-7), and 2-mL aliquots were cultured in 12-well flat-bottomed polystyrene plates (Corning) at 37°C for 4 or 24 h under static conditions. Cells from 1 mL of the culture were harvested by centrifugation at 5,000 × g and 25°C for 10 min and resuspended in 1 mL of PBS. Then, 1 μL of component A (preliminarily diluted 10-fold with DMSO) included in the BacLight RedoxSensor Green Vitality Kit (Invitrogen) was added to the cells and incubated for 10 min at room temperature. Cells were collected by centrifugation, fixed in 4% PFA for 5 min at room temperature, washed twice, and resuspended in 1 mL of PBS. The cell suspensions were diluted 10-fold with DDW, and stained cells were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Cells were flowed at 10 μL/min, and 10,000 events were recorded for each sample.
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