The ability of Sp-LECin to permeate the outer membrane of P. aeruginosa and A. baumannii was measured by the NPN (Sigma, St. Louis, MO, USA) uptake assay as previously described with some modifications [59 (link)]. Briefly, the bacterial cells in the logarithmic phase were harvested, washed, and then suspended in 5 mM HEPES buffer to a final cell density of approximately 1 × 108 CFU/mL. The fluorescent probe NPN dissolved in 95% ethanol (final concentration of 10 μM) was added to the suspension. Subsequently, the above bacterial suspension was aliquoted into a 96-well microplate and incubated with different concentrations of Sp-LECin (from 6 to 48 μM). LL-37 (GL Biochem, Shanghai, China) and polymyxin B (Solarbio, Beijing, China) were used as positive controls for permeabilizing outer membrane of P. aeruginosa and A. baumannii, respectively. Finally, fluorescence was recorded every 2.5 min in a microplate reader (Tecan, Männedorf, Switzerland) with excitation and emission wavelengths of 350 and 420 nm, respectively.
Ll 37
Manufactured by GL Biochem
Sourced in China
LL-37 is a synthetic peptide that corresponds to the C-terminal sequence of the human cathelicidin protein. It functions as an antimicrobial agent and is involved in the innate immune response.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Tecan (1 mentions)
Polymyxin b, by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Ilomastat, by Selleck Chemicals (1 mentions)
E faecalis, by China General Microbiological Culture Collection Center (1 mentions)
S aureus, by China General Microbiological Culture Collection Center (1 mentions)
L monocytogenes, by China General Microbiological Culture Collection Center (1 mentions)
Staphylococcus epidermidis, by China General Microbiological Culture Collection Center (1 mentions)
F graminearum, by China General Microbiological Culture Collection Center (1 mentions)
F solani, by China General Microbiological Culture Collection Center (1 mentions)
P aeruginosa, by China General Microbiological Culture Collection Center (1 mentions)
6 protocols using ll 37
1
Outer Membrane Permeability Assay
2
LL-37 Cytotoxicity Evaluation in NIH3T3 Cells
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All reagents were purchased from Sigma-Aldrich unless otherwise stated. LL-37 (95% purity, LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized by GL Biochem (Shanghai, China). NIH3T3 cells were purchased from the Chinese Academy of Medical Sciences (Tianjin, China). Fetal bovine serum (FBS) and trypsin were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, US); Medium 1640 was purchased from HyClone (GE Healthcare, Chicago, IL, US); Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan).
3
Gliadin and LL-37 Modulate Caco-2 Cells
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Human intestinal epithelial cells Caco‐2 (HTB‐37) were obtained from the American Type Culture Collection (ATCC, VA, USA) and maintained in DMEM containing 1,800 mg l−1 NaHCO3, supplemented with 10% FBS, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin at 37°C in a humidified atmosphere with 5% CO2. LL‐37 (GL biochem, Shanghai, China), a broad‐spectrum matrix metalloproteinase inhibitor Ilomastat (Selleck, Shanghai, China) and PT‐gliadin were dissolved in distilled water. For treatment, cells were pre‐incubated with Ilomastat (5 μM) for 1 h, subsequently stimulated with PT‐gliadin (1 mg ml−1) and LL‐37 (10 µg ml−1) for 3 h. Cells were then collected for measurements. All cell lines were regularly tested negative for mycoplasma contamination.
4
Antimicrobial Peptides and AgNPs Evaluation
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Antimicrobial peptides (AMPs), both LL-37 and LL-31, were purchased from GL Biochem (Shanghai, China). All AMPs were prepared in sterile deionized water at 2.5 mg/ml as stock solution, then aliquoted and stored at −20 °C. One of the AMP stock aliquots was serially diluted by two-fold dilution in the range at a final concentration of 4–512 µg/mL. These AMP solution tubes were kept at −4 °C until use. Tannic acid stabilized AgNPs were given by our collaborator Prime Nanotechnology Co., Ltd. (Bangkok, Thailand) with a stock concentration of 10,000 mg/L. For AgNP solution preparation in our experiments, 1 mg/ml as a stock solution was prepared in sterile deionized water. AgNPs were then serially diluted by two-fold dilution in the range of final concentrations of 4–512 µg/mL, then kept at room temperature until used. Ceftazidime antibiotic (CAZ) was kindly provided by the Melioidosis Research Center, Faculty of Medicine, Khon Kaen University (Khon Kaen, Thailand). The preparation procedure of CAZ was the same method as the preparation method above.
5
Antimicrobial Potency Evaluation
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The strains were purchased from China General Microbiological Culture Collection Center (CGMCC), Beijing, China, including S. aureus (CGMCC 1.2465), L. monocytogenes (CGMCC 1.10753), E. faecalis (CGMCC 1.2135), Enterococcus faecium (CGMCC 1.1310), Staphylococcus epidermidis (CGMCC 1.4260), P. aeruginosa (CGMCC 1.2421), E. coli (CGMCC 1.2389), A. baumannii (CGMCC 1.6769), C. neoformans (CGMCC 2.1563), F. graminearum (CGMCC 3.4521), F. solani (CGMCC 3.5840), and A. niger (CGMCC 3.3160). The bacterial and fungal strains were cultured in nutrient broth (OXOID, UK) agar at 37 °C and yeast extract peptone dextrose (OXOID, UK) agar at 28 °C.
Cells (including murine macrophage RAW 264.7, human embryonic kidney 293 cell (HEK-293T), and hepatocellular carcinoma cell line HepG2) were obtained from the Stem Cell Bank at the Chinese Academy of Sciences (Shanghai, China).
LL-37 was synthesized from GL Biochem (Shanghai, China), and the antibiotic polymyxin B (PMB) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).
Cells (including murine macrophage RAW 264.7, human embryonic kidney 293 cell (HEK-293T), and hepatocellular carcinoma cell line HepG2) were obtained from the Stem Cell Bank at the Chinese Academy of Sciences (Shanghai, China).
LL-37 was synthesized from GL Biochem (Shanghai, China), and the antibiotic polymyxin B (PMB) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).
6
Cramp-mtDNA Complex Antibody Protocol
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LL-37, Cramp, and the corresponding control peptides were purchased from GL Biochem (Shanghai, China). The polyclonal antibody against the Cramp-mtDNA complex was purchased from Abcam (Cambridge, MA).
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