Cells of the bovine mammary epithelial cell line MAC-T were seeded onto 6-well plates (about 1 × 105 cells/well), and then incubated in complete culture medium under standard conditions (37 °C, 5% CO2) in an incubator. The detailed description of the source and culture method of the cells were given in our previous study [19 (link)] and that of Huynh et al. [58 (link)]. When MAC-T cells grew to approximately 70% confluence, they were treated for 24 h with no LPS (CON), 0.1 μg/mL LPS (E. coli O111:B4, Sigma-Aldrich, L2630, St. Louis, MO, USA), and 30 μg/mL PGN (S. aureus, Sigma-Aldrich, 77140, St. Louis, MO, USA) + 30 μg/mL LTA (S. aureus, Sigma-Aldrich, L2515, St. Louis, MO, USA) + 0.1 μg/mL LPS (PLL) in the culture medium, respectively. Previous studies of ours and others have shown that 30 μg/mL PGN or LTA and 0.1 μg/mL LPS can effectively induce the immune responses in BMECs in vitro [18 (link),19 (link),36 (link),39 (link)]. There were six replicates per treatment or group (n = 6) in this study. After 24 h of stimulation, the cells and culture supernatant were collected for further analyses.
L2515
Manufactured by Merck Group
Sourced in United States
The L2515 is a laboratory equipment product manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of the L2515 is to perform precise measurements and data collection, without further interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
L2630, by Merck Group (2 mentions)
Autoitc isothermal titration calorimeter, by Malvern Panalytical (2 mentions)
Lps e coli o111 b4, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
L2880, by Merck Group (1 mentions)
S nhs, by Merck Group (1 mentions)
E1769, by Merck Group (1 mentions)
96 well microtiter plate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase labeled streptavidin, by Agilent Technologies (1 mentions)
5 tetramethylbenzidine liquid substrate, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
L3012, by Merck Group (1 mentions)
P3813, by Merck Group (1 mentions)
10 protocols using l2515
1
Bovine Mammary Epithelial Cell Immune Response
2
bMEC Response to S. aureus Stimuli
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Six cultures of primary bMECs were stimulated with heat-killed S. aureus M5512VL, LTA (10 µg, L2515, Sigma-Aldrich Corp., St. Louis, MO, USA) and S. aureus EVs M5512VL (10 µg) in three different sessions (two cultures per session). The EVs belonged to the same M5512VL bacterial culture, and the dose corresponding to the quantity of protein similarly to what was previously published [17 (link)]. All of the applied treatments were diluted in 1.5 mL DMEM/F12 medium supplemented with Gentamicin and AmpB, except for the control condition (Ctr), in which only medium with Gentamicin and AmpB was added. After 3 or 24 h of incubation, cells were washed twice with PBS, and 300 µL of Trizol™ (Thermo-Fisher Scientific, Waltham, MA, USA) was added per well to detach the cells. Afterwards, the mixture was transferred to a tube and immediately frozen at −80 °C until further analysis.
3
Macrophage Differentiation and Infection
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Following differentiation, human MDMs were seeded (2 × 106/2 ml) in X‐VIVO 15 containing L‐glutamine without gentamicin or phenolred (Biozym/LONZA, #BE02‐061Q) supplemented with 5% (v/v) FCS in 6‐well plates (Greiner Bio‐one). Staphylococcus aureus (for MDM MOI 2; for osteoclasts MOI 10), attenuated S. aureus (heat inactivated for 10 min at 95°C, MOI 2) or 1 μg/ml LTA from S. aureus (L2515, Sigma‐Aldrich) were added at 37°C. Simultaneously, polarization agents were added to MDM as described above. After infection with S. aureus for 2 h, cells were treated with 20 µg/ml recombinant lysostaphin (WAK‐Chemie Medical GmbH, Steinbach, Taunus, Germany) in PBS for 30 min at 37°C to remove extracellular bacteria. Cells were further cultivated without (M0, osteoclasts) or with polarization agents (MIFN‐γ, MIL‐4) for the indicated times (for MDM in X‐VIVO supplemented with 5% (v/v) FCS, 100 U/ml penicillin and 100 µg/ml streptomycin; for osteoclasts in DMEM supplemented with 10% FCS (v/v), 100 U/ml penicillin and 100 µg/ml streptomycin). For measurement of LM, 1 ml of the cell culture medium was collected. In addition, to evoke LM biosynthesis, remaining culture medium was removed, cells were washed with PBS and treated with SACM (1%, 90 min for MDM; 0.5%, 60 min for osteoclasts) in 1 ml PBS plus 1 mM CaCl2 at 37°C.
4
Isothermal Titration Calorimetry of P30-LTA Interaction
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All ITC experiments were performed at 25°C using the AutoITC isothermal titration calorimeter (MicroCal Inc.). The measuring devices and experimental setup details were described previously (64 (link)). The reagents, P30 and LTA (Sigma-Aldrich catalog number L2515-5MG), were dissolved directly in 20 mM HEPES, pH 7.4. The experiment consisted of injecting 10.02 μL (29 injections, 2 μL for the first injection only) of 0.5 mM buffered solution of P30 into the reaction cell, which initially contained 0.05 mM buffered solution of LTA. A background titration, consisting of an identical titrant solution but with the buffer solution in the reaction cell only, was removed from each experimental titration. The LTA solution was injected at 4-min intervals. Each injection lasted 20 s. The stirrer speed was kept constant at 300 rpm. The CaCl2-EDTA titration was performed to check the apparatus, and the results (stoichiometry, K, and ΔH) were compared with those obtained for the same samples (a test kit) at Malvern Instruments Ltd. (Malvern, UK).
5
Binding Kinetics of RC18 to Lipid A and LTA
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The binding kinetics of RC18 to lipid A (L5399, Sigma, Shanghai, China) and lipoteichoic acid (LTA, L2515, Sigma) were measured using the ForteBio Octet Red 96 BLI platform (Sartorius BioAnalytical Instruments, Bohemia, NY, USA). The amine-reactive second-generation (AR2G) biosensors were processed with 1-ethyl-3-(3-dimethylaminopropy) carbodiimide hydrochloride (EDC, E1769, Sigma) and sulfo-N-hydroxysulfosuccinimide (s-NHS, 56485, Sigma). RC18 was diluted with 10 mM sodium phosphate buffer (pH 7.4) at concentrations ranging from 200 to 1200 nM. Lipid A and LTA were loaded onto the activated AR2G biosensors at 30°C for 5 min. Association and disassociation were both carried out for 5 min. To determine the recruitment of peptides to lipopolysaccharide (LPS), AR2G biosensors immobilized with 10 μg/mL LPS (L2880, Sigma) were incubated with 1000 nM HD5 and RC18 for 5 min. The binding thickness was recorded and analysed using ForteBio Data Analysis 7.0 software. The equilibrium dissociation constant (KD) was calculated as the dissociation rate constant (Koff) divided by the association rate constant (Kon).
6
Quantifying Peptide-LPS/LTA Binding
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96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 5 µg/mL of purified LPS (from E. coli O111:B4, Sigma L2630) or LTA (from S. aureus, Sigma L2515) in PBS and then incubated for 2 h at room temperature in blocking solution (20 mM Tris-HCl pH 7.4 plus 0.05% Tween 20 and 1% BSA). Biotin-labeled peptides/proteins (2.5–20 µg/mL) were added and incubated overnight at 4°C in blocking solution. After extensive washing, bound peptides/proteins were detected by the addition of horseradish peroxidase-labeled streptavidin (1:5,000 dilution; DAKO) for 1 h at room temperature. Color was developed by adding 3,3’,5,5’-tetramethylbenzidine liquid substrate (Sigma) and optical density read at 405–620 nm.
7
Antimicrobial Peptide Evaluation
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1 mg/mL of peptides were incubated with an equal volume of 5 mg/mL of the lipopolysaccharide (LPS, Sigma-Aldrich: L3012/L8643) or lipoteichoic acid (LTA, Sigma-Aldrich: L2515) for 30 min. The MICs of peptides treated with LPS or LTA against S. aureus AB94004 were subsequently measured.
8
Antimicrobial Activity of Hp1404 Peptide
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A total of 1 mg/mL of Hp1404 peptide solution was mixed with an equal volume of 5 mg/mL of the lipoteichoic acid (LTA, sigma-aldrich: L2515) or lipopolysaccharide (LPS, sigma-aldrich: L3012) solution. Then, the MICs of Hp1404 treated with LTA or LPS against S. aureus AB94004 were measured. The experiment was verified by three independent trials.
9
Isothermal Titration Calorimetry of P30-LTA Interaction
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All ITC experiments were performed at 25°C using the AutoITC isothermal titration calorimeter (MicroCal Inc.). The measuring devices and experimental setup details were described previously (64 (link)). The reagents, P30 and LTA (Sigma-Aldrich catalog number L2515-5MG), were dissolved directly in 20 mM HEPES, pH 7.4. The experiment consisted of injecting 10.02 μL (29 injections, 2 μL for the first injection only) of 0.5 mM buffered solution of P30 into the reaction cell, which initially contained 0.05 mM buffered solution of LTA. A background titration, consisting of an identical titrant solution but with the buffer solution in the reaction cell only, was removed from each experimental titration. The LTA solution was injected at 4-min intervals. Each injection lasted 20 s. The stirrer speed was kept constant at 300 rpm. The CaCl2-EDTA titration was performed to check the apparatus, and the results (stoichiometry, K, and ΔH) were compared with those obtained for the same samples (a test kit) at Malvern Instruments Ltd. (Malvern, UK).
10
Measuring S. aureus LTA Binding Kinetics
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S. aureus lipoteichoic acid (LTA; L2515, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS) solution (pH=7.4, P3813, Sigma-Aldrich) at concentrations of 0.1, 0.3, 0.5, 1.0 and 2.0 mg/mL. Fivehundred microliters of PBS was added to the sensor cell, and 5 µL of LTA solution was injected into the PBS solution in the sensor cell. The frequency decrease was monitored until 2 h after protein injection.
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