Reported methods were followed to determine the membrane depolarizing and permeabilizing activities of test compounds on M. bovis BCG cultures.23 (link),24 (link) Briefly, M. bovis BCG cultures at mid-log phase were diluted at OD600 0.1 in complete 7H9 broth. Diluted cultures were treated with test compounds at 4× MIC90 for 48 h. At selected time points, aliquots were removed from culture media and tested for changes in membrane potential using BacLight Bacterial Membrane Potential kit (Life Technologies, CA) and membrane permeability using fluorescent probes SYTO-9 and propidium iodide (PI) (Molecular Probes, Invitrogen, MA). RIF at 4× MIC90 (0.08 μM) was used as the negative control for both assays. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 100 μM and a detergent SDS (sodium dodecyl sulfate) at 5% (v/v) were used as the positive controls in membrane potential and permeability tests, respectively. In the membrane permeability assay, drug-treated samples were normalized against drug-free cultures (0% permeabilization) and SDS-treated cultures (100% permeabilization) to give % permeabilization.23 (link)
Baclight bacterial membrane potential kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BacLight Bacterial Membrane Potential Kit is a fluorescence-based assay designed to measure the membrane potential of bacterial cells. The kit utilizes a dual-fluorescence probe that reports on the relative membrane potential of the cells.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (6 mentions)
Cflow, by BD (4 mentions)
Biosciences accuri c6 flow cytometer, by BD (3 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (3 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
S3e cell sorter, by Bio-Rad (2 mentions)
Accuri c6, by BD (2 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Synergy 2 microplate reader, by Agilent Technologies (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Bactiter glo kit, by Promega (1 mentions)
Guava expressplus, by Merck Group (1 mentions)
Guava flow cytometer, by Merck Group (1 mentions)
Synergy ht multi mode microplate reader, by Agilent Technologies (1 mentions)
Facsaria, by BD (1 mentions)
Xmark microplate absorbance spectrophotometer, by Bio-Rad (1 mentions)
Macsquant, by Miltenyi Biotec (1 mentions)
Total reactive oxygen species assay kit, by Thermo Fisher Scientific (1 mentions)
68 protocols using baclight bacterial membrane potential kit
1
Mycobacterium Membrane Activity Assay
2
Membrane Potential Assessment by Flow Cytometry
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Membrane potential was assessed using a flow cytometry assay based on the BacLight bacterial membrane potential kit (Life Technologies). Cells from overnight cultures were inoculated in 10 ml TSB in 100-ml Erlenmeyer flasks and grown to an optical density at 600 nm (OD600) of 0.2. Fifteen microliters of culture was transferred to 1 ml filtered PBS. To each cell solution, 10 μl of the fluorescent membrane potential indicator dye DiOC2 (3 (link)) was added and cells were stained for 5 min at room temperature. Data were recorded on a BD Biosciences Accuri C6 flow cytometer (Becton, Dickinson and Company), with emission filters suitable for detecting red and green fluorescence. Settings on the flow cytometer were as follows: 50,000 recorded events at a forward scatter (FSC) threshold of 15,000 and medium flow rate. Gating of the stained cell population and analysis of flow cytometry data were performed in CFlow (BD Accuri). As an indicator of membrane potential, the ratio of red to green fluorescence intensity was calculated. The assay was verified with the NTML mutant containing a transposon insertion in menD (NE1345), which displays depolarization of the membrane (45 (link)).
3
Membrane Potential Profiling of E. coli
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The procedure for studying membrane potential changes in prodrug treated E. coli was identical to that used for the Live/Dead assay above with the following exceptions. The BacLight™ Bacterial Membrane Potential Kit (Lifetech Cat # B34950) was used in this case. 10 μl of Component A (3 mM DiOC2) was used to stain the bacterial samples for 30 min. at room temperature. TPP or component B (CCCP) treated bacteria were used as negative or positive controls respectively and to gate prodrug treated samples. With intact membrane potential the DiOC2 dye form tetramers within bacteria that fluoresce at 630 nm (red). Loss of membrane potential leads to dimer formation that fluoresce at 530 nm (green). Bacteria were analyzed similarly by both flowcytometry and microscopy.
4
Membrane Potential Assay via Flow Cytometry
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Assessment of variations in membrane potential was estimated using a flow cytometry assay based on the BacLight Bacterial Membrane Potential Kit (LifeTechnologies). Cells from ON cultures were inoculated in 30 ml TSB in 300 ml Erlenmeyer flasks and grown to an OD600 of 0.2. Fifteen microliter culture was transferred to 1 ml filtered phosphate-buffered saline (PBS). To each cell solution 10 μl of fluorescent membrane potential indicator dye, DiOC2(3), was added and cells were stained for 30 min at room temperature. Data was recorded on a BD Biosciences Accuri C6 flow cytometer (Becton, Dickinson and Company), with emission filters suitable for detecting red and green fluorescence. Settings on the flow cytometer were as follows: 50000 recorded events at a FSC threshold of 15000 and medium flow rate. Gating of stained cell population and analysis of flow cytometry data were performed in CFlow® (BD Accuri). As an indicator of membrane potential the ratio of red to green fluorescence intensity was calculated. The assay was verified using the two SCV strains with transposon insertions in hemB (NE1845) and menD (NE1345).
5
Analyzing Membrane Potential Changes in S. pneumoniae
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To detect changes in the membrane potential of S. pneumoniae TIGR4 when treated with Carolacton, the bacteria were stained with the fluorescent dye 3,3-Diethyloxacarbocyanine iodide (DiOC2(3)) (BacLight Bacterial Membrane Potential Kit, Life Technologies). For staining with DiOC2(3), cells were diluted from culture to approx. 107 cells/ml and stained with 30 μM DiOC2(3) in the dark for 30 min. In order to assess bacterial membrane integrity, cells were separately co-stained with the fluorescent dyes Syto 9 and propidium iodide (PI) using the LIVE/DEAD BacLight Bacterial Viability Kit (Life Technologies).
For Syto 9/PI staining, cells from a culture grown with 0.25 μg/ml Carolacton and from a control were harvested by centrifugation and washed in 0.85% (w/v) NaCl. Approx. 5 × 106 cells/ml were stained with an equal volume 2× staining solution (10 μM Syto 9, 60 μM PI) at RT in the dark for 30 min. Heat-inactivated cells were used as a control in both staining experiments. Heat-killing of cells was achieved by incubation at 80 °C and shaking (1,400 rpm) for 30 min.
For Syto 9/PI staining, cells from a culture grown with 0.25 μg/ml Carolacton and from a control were harvested by centrifugation and washed in 0.85% (w/v) NaCl. Approx. 5 × 106 cells/ml were stained with an equal volume 2× staining solution (10 μM Syto 9, 60 μM PI) at RT in the dark for 30 min. Heat-inactivated cells were used as a control in both staining experiments. Heat-killing of cells was achieved by incubation at 80 °C and shaking (1,400 rpm) for 30 min.
6
Salmonella Membrane Potential Assay
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Arginine was added into the M9 medium of Gentamicin-resistant Salmonella. The mixture was then incubated at 37°C for 8 h. After incubation, 1 mL of bacterial solution was removed and centrifuge at 20,000 × g for 5 min at −10°C. The supernatant was discarded. The precipitate was washed twice with PBS and then diluted to approximately 1 × 106 CFU/mL with PBS. The membrane potential was examined using a BacLight Bacterial Membrane Potential Kit (Life Technologies, Carlsbad, CA, United States) as previously described (Zhou et al., 2022 (link)).
7
Membrane Potential of Apramycin-Resistant Salmonella
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Different exogenous substances were added into the culture medium of Apramycin-resistant Salmonella (Apr-R-ATCC13312 and Apr-R-CICC21484), which were then cultured at 37°C for 8h. Then, exactly 1ml of the bacterial solution was removed and centrifuged at 20000g for 5min at −10°C. The supernatant was discarded; the precipitate was washed twice with PBS and then diluted using PBS to approximately 1×106 cells per ml. Then, the membrane potential was measured using a BacLight Bacterial Membrane Potential Kit (Life Technologies, Carlsbad, CA, United States) according to the manufacturer’s instructions.
8
Membrane Potential Determination in MRSA
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The membrane potential of the study strains was determined using a BacLight bacterial membrane potential kit (Life Technologies, Carlsbad, CA, USA) (17 (link)). In brief, MRSA cultures were adjusted to an OD600 of 0.4 in 1 mL of filtered PBS, mixed with 10 μL of 3 mM 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)] fluorescent dye, and incubated at room temperature for 30 min. In parallel, a control with an additional 10 μL of 500 μM proton motive force inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was prepared for each sample. The fluorescent signals were determined at an excitation wavelength of 485 nm and an emission wavelength of 528 nm (green fluorescence) and an excitation wavelength of 485 nm and an emission wavelength of 590 nm (red fluorescence) using a BioTek Synergy 2 microplate reader (BioTek Instruments, Winooski, VT, USA). The ratio between red fluorescence and green fluorescence was calculated to indicate membrane potential (17 (link)).
9
Bacterial Membrane Potential Analysis
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The membrane potential was examined using a BacLight Bacterial Membrane Potential Kit (Life Technologies, Carlsbad, CA, United Statessss) as previously described (Yong et al., 2021 (link)).
10
Membrane Potential of M. bovis BCG
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