Disruption of the outer bacterial membrane of P. aeruginosa was measured using the fluorescent probe 1-N-phenylnaphthylamine (NPN; Merck). Late-exponential-phase cultures of P. aeruginosa were washed twice and adjusted to an OD600 of 0.5 in 5 mM HEPES buffer. In the wells of a black microtiter plate (ThermoScientific), adjusted cultures were combined with NPN (final concentration, 10 μM) along with either no treatment (buffer only), 50 mg/L GA, 2 mg/L CST, 4 mg/L TOB, or 16 mg/L LL-37 (Merck), to a final volume of 200 μL. Agent concentrations were chosen based on EUCAST breakpoint concentrations (for CST and TOB) or previously published active concentrations (GA [18 (link)] and LL-37 [73 (link)]). Each experiment consisted of triplicate wells of each condition. Fluorescence was measured in a plate reader at excitation of 355 nm, emission at 460 nm every 30 s for 15 min. NPN uptake factor was calculated as follows: [(fluorescence of sample with NPN) − (fluorescence of sample without NPN)]/[(fluorescence of buffer with NPN) − (fluorescence of buffer without NPN)].
Ll 37
Manufactured by Merck Group
Sourced in United States, India
LL-37 is a polypeptide that is part of the cathelicidin family of antimicrobial peptides. It serves as a core component in certain laboratory equipment and reagents. The primary function of LL-37 in these applications is to provide an effective antimicrobial agent to help maintain the integrity and sterility of the laboratory environment and samples.
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Lab products found in correlation
Cecropin, by Merck Group (2 mentions)
Polymyxin b, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Primescripttm rt reagent kit, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Mannuronic acid, by Merck Group (1 mentions)
Chitosan, by MP Biomedicals (1 mentions)
Hyaluronic acid, by Merck Group (1 mentions)
Dextran, by Merck Group (1 mentions)
Phalloidin fluorescein isothiocyanate, by Merck Group (1 mentions)
Ag1478, by Merck Group (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Vancomycin, by Merck Group (1 mentions)
Polymyxin b, by HiMedia (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Lsm 700, by Zeiss (1 mentions)
19 protocols using ll 37
1
Membrane Disruption Assay for P. aeruginosa
2
Cathelicidin Antimicrobial Peptide Assay
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Cathelicidins represent prototypical CAMPs and are characterized the key elements in the host defense mechanisms within epithelial cells, intestinal mucosa, and skin. Human cathelicidin (LL-37) [26 (link)] and canine cathelicidin (K9CATH) [27 (link)] were synthesized at GL Biochen (China) with purity > 95%.
Standard MIC testing in nutrient broth, such as Mueller Hinton broth, may inhibit the bactericidal activity of CAMPs [28 (link)]. Thus, in vitro susceptibility assays with LL-37 and K9CATH were carried out as previously described using the 2 h microdilution method in RPMI-1640 medium (Sigma) supplemented with 5% Luria-Bertani broth. Briefly, the assays were performed with LL-37 (1 μg/mL) and K9CATH (3 μg/mL) using an initial S. schleiferi cell inoculum of 5 × 103 CFUs. These LL-37 and K9CATH concentrations were determined through extensive preliminary assays displaying their inability to completely kill the initial inoculum of the S. schleiferi cells over the 2-h assay duration. Data represent the relative percentage of surviving CFUs (± standard deviation [SD]) of CAMP-treated versus untreated cells. At least three independent experiments were performed for each cathelicidin peptides.
Standard MIC testing in nutrient broth, such as Mueller Hinton broth, may inhibit the bactericidal activity of CAMPs [28 (link)]. Thus, in vitro susceptibility assays with LL-37 and K9CATH were carried out as previously described using the 2 h microdilution method in RPMI-1640 medium (Sigma) supplemented with 5% Luria-Bertani broth. Briefly, the assays were performed with LL-37 (1 μg/mL) and K9CATH (3 μg/mL) using an initial S. schleiferi cell inoculum of 5 × 103 CFUs. These LL-37 and K9CATH concentrations were determined through extensive preliminary assays displaying their inability to completely kill the initial inoculum of the S. schleiferi cells over the 2-h assay duration. Data represent the relative percentage of surviving CFUs (± standard deviation [SD]) of CAMP-treated versus untreated cells. At least three independent experiments were performed for each cathelicidin peptides.
3
Ceragenins and AMPs against P. aeruginosa
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Ceragenins (CSA-13, CSA-44, CSA-90, CSA-131, CSA-131-poloxamer (CSA-131P), CSA-138, CSA-142, CSA-144 and CSA-192) were synthesized from cholic acid. AMPs (magainin, cecropin, and LL-37) and Pluronic® F127 used to prepare CSA-131-poloxamer were obtained from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of ceragenins and AMPs were prepared by dissolving the materials in distilled water and storing at −20 °C for a maximum of one month. CSA-131 was combined with 5% Pluronic F-127 to form CSA-131-poloxamer and stored at room temperature. P. aeruginosa-GFP (ATCC® 10145GFP™) containing a multicopy vector encoding the green fluorescent was used in the study.
4
Antibacterial Peptide Gene Expression Assay
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For the antibacterial peptide assay, the bacteria were cultured in M9 medium with or without LL-37 (Sigma) and incubated at 37 °C for 1–2 h. Blood or bacteria were pelleted by centrifugation. RNA samples were isolated using TRIzol (Invitrogen), reverse transcribed using the PrimeScriptTM RT reagent Kit (TaKaRa, Kusatsu, Japan), and processed for qRT–PCR. Each qRT–PCR was reacted in Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA). The fold-change in the target gene relative to the housekeeping gene dnaE was determined using the 2−ΔΔCt method. At least three biological replicates were used for each qRT–PCR analysis.
5
Antimicrobial Susceptibility Testing Protocol
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Bacterial strains were grown to mid-exponential phase (approximately an optical density at 600 nm [OD600] of ~0.5 or 2 × 108 CFU/ml). For H2O2 killing assays, bacteria were mixed 1:1 with H2O2 diluted in LBNS and incubated for 1 h at 37°C, followed by plating for CFU per milliliter on PIA. Data were expressed as log fold killing relative to the no-treatment condition. For the LL-37 (Sigma) killing assays, bacteria at the exponential phase were pelleted and resuspended in sodium phosphate buffer (SPB) at pH 6.4. Bacteria were mixed 1:1 with LL-37 diluted in SPB and incubated 1 h at 37°C, followed by plating for CFU per milliliter on either PIA or LA. For experiments in which mono- or polysaccharides were added exogenously, seaweed alginate (Sigma), hyaluronic acid (Sigma), dextran (Sigma), chitosan (MP Biomedicals), mannuronic acid (Sigma), and guluronic acid (Carbosynth, Compton, United Kingdom) were obtained commercially. P. aeruginosa alginate was purified as described below.
6
Molecular Probes and Inhibitors Protocol
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Hoechst 33258, 3(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium bromide (MTT), AG1478, mitomycin C; phalloidin-fluorescein isothiocyanate, LL-37 were from Sigma-Aldrich (St. Luis, MO); GM6001 was from Calbiochem (Merk Millipore, Germany).
7
LL-37 Modulates Vancomycin Efficacy
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Staphylococcus aureus FPR3757 was diluted to 105 CFU/ml in MH or MHII media and incubated for 1 h (200 rpm, 37 °C) with either no, 5, 10 or 20 μg/ml LL-37 (Sigma) followed by addition of vancomycin (Sigma). The minimum inhibitory concentration (MIC) was determined in a 96-well microtiter plates (200 rpm, 37 °C, 24 h using technical duplicates and biological triplicates) by visual inspection. Similar approach was used for determining MIC for LL-37 with increasing LL-37 concentrations and no addition of vancomycin.
8
Antimicrobial Peptide Susceptibility Assay
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AMP susceptibility human neutrophil defensin-1 (hNP-1) (AnaSpec Incorporated, CA, USA) and LL-37 (Sigma) susceptibility assays were performed as described previously (Duggan et al., 2020 (link)). Briefly, overnight cultures were normalised to an OD600nm of 0.1 and incubated with 5 μg/ml of hNP-1 or LL-37 for 2 hr at 37°C. Serial dilutions were plated on TSA to determine CFUs. The same number of bacterial cells inoculated into PBS, diluted, and plated acted as a control. Survival was determined as the percentage of CFU on exposure to either LL-37 or HNP-1 relative to the PBS control. Relative survival was determined through normalisation to JE2.
9
Antimicrobial Peptide Sensitivity Assays
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The AMP sensitivity assays were performed as previously described [27 ]. Briefly, overnight cultures of WT, Δ1538, and the complemented strain cΔ1538 were subcultured (1:100) in LB medium at 37°C and 150 rpm until 0.4 OD.600 (log phase). Strains were then diluted with 1X phosphate-buffered saline (PBS) to obtain 108 cfu/mL. Each strain suspension was then incubated at 37°C for 1 h with 1 µg/mL AMP, 1 µg/mL polymyxin B (Himedia, India), and 10 µg/mL LL-37 (Sigma). Counts for the number of surviving bacteria were obtained by serial dilutions and plating on LB agar. The bacterial survival percentage was obtained by dividing the colony-forming units (CFU) post-AMP treatment against the CFU pre-AMP treatment, with WT survival normalized to 100%.
10
Antimicrobial Peptide Susceptibility Assay
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Overnight grown culture of wild-type and mutant strains was subcultured for 4 h. Cells were washed with PBS and diluted to approximately 108 CFU/ml. A 50 µl aliquot of bacterial suspension was incubated at 37 °C for 1 h with or without AMPs, polymyxin B; 1 µg/ml (Sigma), protamine; 5 µg/ml (Himedia, India), LL37; 10 µg/ml (Sigma), cecropin; 2 µg/ml (Sigma) in PBS. Subsequently, samples were serially diluted and plated on LB agar plates to obtain the number of surviving bacteria. The survival efficiency was calculated as percent ratio of the CFU obtained from AMP treated culture versus untreated culture. Experiment was repeated thrice in triplicates.
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