Bacteria were treated as described in the section above (“Cultivation and preparation of bacteria”). Chips were incubated with the BacTiter-Glo™ (Promega, UK) mixture in 96 well plates, as recommended by the manufacturer. After the incubation time, the solution was transferred into a white 96 well plate (Costar, UK) and luminescence was measured using a Tecan infinite 200 pro microplate reader. Luminescence values were normalised to the background intensity measured in adjacent empty wells. Data was analysed and plotted using the software package R. This experiment was performed twice, each with two technical replicates under identical conditions (n = 4).
Bactiter glo
Manufactured by Promega
Sourced in United States
The BacTiter-Glo Microbial Cell Viability Assay is a luminescent assay that quantifies the amount of ATP present in a sample, which is an indicator of metabolically active cells. The assay uses a proprietary thermostable luciferase and a stabilized luciferin substrate to generate a luminescent signal proportional to the amount of ATP present in the cells.
Automatically generated - may contain errors
Lab products found in correlation
Clariostar, by BMG Labtech (2 mentions)
A5508, by Merck Group (2 mentions)
Enspire multimode reader, by PerkinElmer (2 mentions)
White 96 well plate, by Corning (1 mentions)
Infinite 200 pro microplate reader, by Tecan (1 mentions)
Envision multimodal plate reader, by PerkinElmer (1 mentions)
Victor3, by PerkinElmer (1 mentions)
Bactiter glo kit, by Promega (1 mentions)
Spectramax i3x plate reader, by Molecular Devices (1 mentions)
Flexstation 3 microplate reader, by Molecular Devices (1 mentions)
Victor3 plate reader, by PerkinElmer (1 mentions)
Filtermax f5, by Molecular Devices (1 mentions)
Fluoroskan ascent fl plate reader, by Thermo Fisher Scientific (1 mentions)
Spectramax i3, by Molecular Devices (1 mentions)
Glutaraldehyde solution, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Alginic acid sodium salt, by Merck Group (1 mentions)
Wst 1 assay, by Roche (1 mentions)
Tristar lb 941, by Berthold Technologies (1 mentions)
47 protocols using bactiter glo
1
Bacterial Viability Assay
2
Gametocyte Enrichment and ATP-Based Viability Assay
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Mature gametocytes, predominantly stage V, were enriched using density gradient centrifugation and magnetic separation. For density gradient separation, gametocytes were pelleted and resuspended in 10 ml of glucose-rich complete medium, loaded onto 5 ml preheated (37 °C) NycoPrep™ 1.077 cushions (Axis-Shield) and centrifuged at 800xg for 20 min at 37 °C. The gametocyte-containing bands were collected, concentrated by centrifugation and the pellet resuspended in 5 ml glucose-rich complete medium. This was loaded on equilibrated LS-columns for magnetic separation in a MidiMACS magnetic system (Miltenyi Biotec) to purify and enrich the gametocytes, which were counted in a Neubauer chamber. Drug dilutions were placed in triplicate in 96-well plates. Approximately 30,000 gametocytes in glucose-rich complete medium were added to each well in a final volume of 100 μl and the plates incubated for 48 h in a humidified gas chamber (90 % N2, 5 % O2, and 5 % CO2) at 37 °C. Subsequently, the BacTiter-Glo™ assay (Promega) was performed according to the manufacturer’s instructions at room temperature in the dark, with assay substrate incubation for 10 min and shaking for the first two minutes, to detect ATP levels. Bioluminescence [17 (link), 21 (link), 32 (link)] was detected at an integration constant of 0.5 s with the GloMax®-Multi + Detection System with Instinct® Software.
3
ATP-based Antimicrobial Screening Assay
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While the resazurin-based method was a reliable way to test the phenotypic activity of antitubercular compounds, it was unfortunately unsuitable for HTS campaigns given the low signal-to-noise ratio and the frequent interference of fluorescent compounds. As an alternative to a resazurin-based readout, we used a commercially available system based on ATP measurement (BacTiter-Glo, Promega). This assay measured the effect of the compounds on bacterial growth by determining the amount of ATP per well, which is proportional to the number of living bacteria. The reagent caused bacterial cell lysis and generated a luminescent signal proportional to the amount of ATP present and thus to the number of viable cells in culture. The assay relied on the activity of a thermostable luciferase and on the properties of a buffer formulation for extracting ATP from bacteria.
4
Borrelia Drug Susceptibility Assay
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About 200 μL of 106/mL Borrelia strains were grown in white opaque bottom 96-well plates with drugs at respective concentrations ranging from 0 μM to 250 μM. After 96 hours, 100 μL of culture was taken from each well and mixed with 100 μL of Bac Titer-Glo® reagent (Promega, Madison, WI, USA). Then, the assay was performed according to the manufacturer’s instructions. Luminescence was measured on a Flex Station 3 micro plate reader at an integration time of 500 milliseconds.
5
Antibacterial Assay of Huanamycin Analogs
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TABLE 1 Antibacterial Assays of compounds 2 to 10 Compound No. MIC (μg/mL) Huanamycin A 8 μg/mL 2 >32 μg/mL 3 16 μg/mL 4 16 μg/mL 5 >32 μg/mL 6 >32 μg/mL 7 16 μg/mL 8 >32 μg/mL 9 Not determined 10 8 μg/mL
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Example 9
The minimum inhibitory concentrations (MICs) were determined using the Promega Bac Titer-Glo microbial cell viability assay, which measures cell viability by quantitation of ATP present, an indicator of metabolic activity of cells. The assays were carried out using Salmonella enterica strain AMC (ATCC #6539), Inocula of S. enterica were prepared from 12-h broth cultures grown in Mueller Hinton broth and the suspensions were then adjusted to a turbidity of 0.5 McFarland. Assays were conducted in a 96-well plate using growth media with an inoculum of ˜5˜104 CFU/mL using the suggested protocols. Bacterial cells were treated with hunanamycin analogs for 24 hours at ranges from 0.4 to 40 μg/mL and ciprofloxacin as a control ranging from 0.03 to 10 μg/mL. The OD600 was measured using an Envision multi-modal plate reader (Perkin-Elmer, Inc.). The antibiotic assays were performed in Prof. John B. MacMillan's lab, Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, USA.
6
Targeted Metabolite Analysis in S. pneumoniae
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For metabolites that were not detected using metabolomics, targeted analyses using commercially available kits were undertaken for the detection of ethanol (Megazyme), acetate (Biovision), ATP (BacTiter-Glo; Promega) and NAD(P)H/NAD(P) (Abcam). All assays were carried out according to the manufacturer’s instructions. In brief, S. pneumoniae were grown in 7 mL cultures of CDM ± 30 µM Cd2+ as previously described in a minimum of biological triplicate. Cells were harvested via centrifugation at 7000 × g for 7 min at 4 °C and resuspended in assay-appropriate buffer. All statistical analyses were conducted using a two-tailed, unpaired t-test (GraphPad Prism).
7
Metabolite Quantification in M. smegmatis
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The ATP concentration was measured by using a BacTiter-Glo (Promega, Madison, WI) luminescence kit. The glucose-6-phosphate (G6P) concentration and the NADP/NADPH ratio were respectively measured with an Amplite (AAT Bioquest, Sunnyvale, CA) colorimetric G6P assay and colorimetric NADP/NADPH ratio assay kits. M. smegmatis grown for 24 h in 7H9T–0.02% glucose was washed once with PBS. The pellets were resuspended in PBS and lysed by bead beating. Lysates were normalized by total protein concentration using a BCA protein assay kit (Pierce, Rockford, IL) and then processed according to the manufacturer’s protocol.
8
Axenic LdBOB Cell Quantification
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Serial dilutions of LdBOB cells with defined cell concentrations were made. Each of the resulting concentrations was dispensed into at least 24 wells of a 384-well plate (50 μl and 25 μl per well for determining the LoD of the historic and novel axenic assay respectively), the rest of the wells contained media only (blank). The read-out for the historic axenic assay was performed by adding resazurin at 0.05 mM final concentration followed by incubation for 4 h at 37°C and 5% CO2. Fluorescence intensity was then measured using a Perkin Elmer Victor 3 plate-reader (excitation 528 nm, emission 590 nm). The read-out for the novel axenic assay was carried out by adding BacTiter-Glo (Promega) (volume equal to culture media volume) to each well and the luminescence was immediately read in a Victor 3 plate-reader. A linear regression was fitted and the LoD was derived as the number of cells equal to the mean signal of the blank wells plus 3 times the standard deviation. The values were determined from 4 independent experiments LoDs are reported as LoD +/- Standard Deviation (StDev).
9
Quantifying ATP Levels in AgNP-Treated Bacteria
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The quantitative determination of ATP in the bacterial cells, as given above, after treatment with AgNPs (concentration range 0.125–512 μg/ml) was determined using BacTiter-Glo™ kit (Promega, Madison, WI, United States) following the manufacturer’s instruction. The test was performed in triplicate, in sterile 96-well plates. Each well contained sterile distilled water as a diluent, glucose (0.2% final concentration), AgNPs at the specified final concentration, as indicated above, and inoculum of bacteria (1.2 × 107 CFU/ml). The total volume of the test sample in each well was 100 μl. The appropriate controls were also maintained as follows:
Plates were incubated for 4 h at 37°C. Subsequently, the test samples (50 μl) were transferred to 96-well opaque plates and mixed with buffer containing substrate (luciferin, BacTiter-Glo™, Promega, Madison, WI, United States) and thermostable luciferase (Ultra-Glo™ Recombinant Lucyferase, Promega, Madison, WI, United States) in a ratio of 1:1 (v/v). Plates were incubated for 5 min at room temperature and luminescence of samples measured at 600 nm by using Synergy HT BioTek Reader (United States). The results were expressed as the percent of inhibition of ATP synthesis in bacterial cells treated with AgNPs when compared with control (untreated cells).
Plates were incubated for 4 h at 37°C. Subsequently, the test samples (50 μl) were transferred to 96-well opaque plates and mixed with buffer containing substrate (luciferin, BacTiter-Glo™, Promega, Madison, WI, United States) and thermostable luciferase (Ultra-Glo™ Recombinant Lucyferase, Promega, Madison, WI, United States) in a ratio of 1:1 (v/v). Plates were incubated for 5 min at room temperature and luminescence of samples measured at 600 nm by using Synergy HT BioTek Reader (United States). The results were expressed as the percent of inhibition of ATP synthesis in bacterial cells treated with AgNPs when compared with control (untreated cells).
10
Bacterial Viability Assay with BacTiter Glo
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Mid-log phase bacteria were exposed to drug for 24 hours at 37°C then 25μL of bacteria was mixed with 25μL of BacTiter Glo™ reagent (Promega) as per manufacturer’s instructions and as previously described (104 (link)). Samples were mixed by orbital shaking at 100 rpm for 10 minutes at room temperature and then luminescence was measured in a black half-area 96 well plate using a plate reader (CLARIOstar, BMG LabTech) at 545-50 nm emission.
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