Exponential cultures in LB broth were prepared, and samples containing around 3 × 10² colony-forming-units (CFU) were transferred to polypropylene microtiter plates (Soria Genlab) containing known amounts of sodium deoxycholate (DOC) (Sigma Aldrich), the archetypal and most abundant bile salt72 (link). Growth was visually monitored after 12–36 h. Similar protocols were used for determination of MICs of polymyxin B sulfate salt (Sigma Aldrich) and hydrogen peroxide. The Student’s t test was used to determine whether the differences in MIC values were significant.
Polymyxin b sulfate salt
Manufactured by Merck Group
Sourced in United States
Polymyxin B sulfate salt is a chemical compound used as a laboratory reagent. It is a mixture of cyclic polypeptide antibiotics derived from the bacterium Paenibacillus polymyxa. Polymyxin B sulfate salt is used in research and scientific applications, but its specific functions and intended uses are not provided in this factual description.
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Lab products found in correlation
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Sodium deoxycholate doc, by Merck Group (1 mentions)
2 octen 1 ylsuccinic anhydride, by Merck Group (1 mentions)
Goat anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti iκbα antibody, by Cell Signaling Technology (1 mentions)
Hrp labeled anti mouse igg antibody, by Cell Signaling Technology (1 mentions)
Hrp labeled anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Tak 242, by ChemScene (1 mentions)
Lps from escherichia coli 026 b6, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline solution, by Thermo Fisher Scientific (1 mentions)
Calcein am, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
14 protocols using polymyxin b sulfate salt
1
Microdilution Assay of Antimicrobial Agents
2
Anti-inflammatory Signaling Pathway Modulation
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2-Octen-1-ylsuccinic anhydride, RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, polymyxin B sulfate salt, and LPS from Escherichia coli 026/B6 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA) and Biolegend (San Diego, CA, USA), respectively. Goat anti-actin antibody and anti-goat IgG antibody labeled with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-IκBα antibody, HRP-labeled anti-mouse IgG antibody, HRP-labeled anti-rabbit IgG antibody, and rabbit antibodies against histone H3, NF-κB p65, ERK1/2, phosphorylated ERK1/2, JNK, phosphorylated JNK, p38 MAPK, and phosphorylated p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA). TAK-242 was purchased from Chemscene (Monmouth Junction, NJ, USA).
3
Peptide-based Nanoparticles for Cell Studies
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Polymyxin B sulfate salt, calcein AM, ethidium homodimer-1 (EthD-1), dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), and hydrochloric acid (HCl, 37 wt%) were acquired from Sigma-Aldrich and used directly. Fluorescein isothiocyanate was provided by Thermo Fisher and used as received. Two amphiphilic peptides (Nap-Gly-Phe-Phe-Phe-Gly-Val-Asp-OH and Nap-Gly-Phe-Phe-Phe-Gly-Val-CONH2) were designed according to our previously studies (Xu et al., 2011 (link), 2012 (link)) and synthesized by GL Biochem Ltd. (Shanghai, China). Phosphate buffered saline (PBS) solution, Dulbecco’s Modified Eagle’s Medium (DMEM), luria broth (LB) medium, fetal bovine serum (FBS), penicillin-streptomycin, trypsin, and were provided by GIBCO Invitrogen Co. All other reagents and solvents were of analytical grade and used directly.
4
Antibody Reagents for Immune Analyses
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Antibodies against CD63 (unconjugated, mouse-anti-human clone H5C6) and CD81 (PE conjugated, mouse-anti-human clone JS-81) were obtained from BD (BD Biosciences, Franklin Lakes, NJ, USA). Purified lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma (St. Louis, MO, USA). Pam3CSK4 was obtained from InvivoGen (InvivoGen, San Diego, CA, USA). Polymyxin B sulfate salt was from Sigma (St. Louis, MO, USA).
5
Graphene Oxide Synthesis and Characterization
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GO was synthesized following a modified Hummers’ method (Morimoto et al., 2016 (link)) and obtained as an aqueous dispersion with a concentration of 3 mg/mL. The solvents were obtained from commercial suppliers and used without purification. Water was purified using a Millipore MilliQ® filter system equipped with the free endotoxin Polisseur Biopak. When stated, the suspensions were sonicated in a water bath Elmasonic P sonicator with settings at 20 W and 40 kHz. MWCO 12,000-14,000 Da dialysis membranes were purchased from Spectrum Laboratories, Inc. Polymyxin B sulfate salt (mixture of polymyxin B1 and B2) and 2,2′-(ethylenedioxy)bis(ethylamine) were purchased from Sigma Aldrich. Depending on the types of containers employed, either 50 mL conical tubes or 1.5 mL microtubes, two centrifuges (Eppendorf Centrifuge 5804R or Eppendorf Centrifuge 5415R) were used.
6
Isolation and Culture of Macrophages from Mice
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BMDMs and alveolar macrophages were isolated and cultured under sterile techniques as described in detail by McMahan et al23 (link) and Manicone et al.26 (link) Briefly, for BMDMs, marrow from femurs and tibias of wild-type and Mmp10−/− mice was recovered by brief centrifugation. Red blood cells were removed by adding lysis buffer (eBio-science, San Diego CA, USA). The remaining cells were suspended in Mac medium (RPMI 1640 with 10% fetal bovine serum and 20% medium conditioned by L929 cells as a source of CSF-1) and plated at 1.5×106 cells/10 cm plate. Mac medium was replaced on days 3 and 6. Between days 7 and 10, plates were washed with PBS to remove nonadherent or dead cells. The macrophages were then removed from the plate using 5 mM ETDA in PBS, and cell counts and viability analysis were determined. Alveolar macrophages were isolated from BAL by centrifugation, pooled in pairs, and treated with 100 µg/mL of MWCNTs in low serum medium (RPMI 1640 + 2% FBS) for 2 or 24 h. Polymyxin B sulfate salt (10 µg/mL; Sigma-Aldrich, St Louis, MO, USA) was included in experimental treatments to control for potential lipopolysaccharide contamination.
7
Immune Response Cell Culture Protocols
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Minimum Essential Medium (MEM, Gibco®), Dulbecco’s modified Eagle’s medium (DMEM, Gibco®), L-glutamine, trypsin, fetal bovine serum (FBS, Gibco®), ortho-phenylenediamine dihydrochloride, isopropyl-1-thio-β-D-galactopyranoside (IPTG), Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, Tween-20, penicillin, and streptomycin were purchased from Thermo Fisher Scientific, (Boston, MA, USA). Bovine serum albumin (BSA), lipopolysaccharides (LPS) from E. coli 026/B6, Triton X-114, anti-mouse IgG antibody labelled with horseradish peroxidase, proteinase K, polymyxin B sulfate salt, collagen Type I (purified from rat tail), collagen Type IV and laminin (purified from basement membrane of Engelbreth-Holm-Swarm mouse sarcoma), elastin (purified from bovine neck ligament), fibronectin, plasminogen and fibrinogen (purified from human plasm) were products of Sigma-Aldrich (St. Louis, MO, USA). Human FH and C4 were purchased from Complement Technology (Texas, USA).
8
Splenocyte Cytokine Secretion Analysis
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Spleens were pooled by pairs (5 pools group−1) and were mechanically homogenized (GentlMACS Dissociator, Miltenyi Biotec, USA). After lysis of red blood cells with lysing buffer (Red Blood Cell Lysing Buffer Hybrid‐Max, Sigma‐Aldrich), splenocytes were resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM l ‐glutamine, 100 U penicillin and 100 mg mL−1 streptomycin. Cells (106 cells well−1) were incubated for 60 h at 37 °C (5% CO2) in 96‐well culture plates in the presence of purified SFS protein (20 µg mL−1) or PBS. Polymyxin B sulfate salt (50 µg mL−1, Sigma‐Aldrich) was added to each activator for endotoxin neutralization. Cytokines secretion (IL‐4, IL‐5, IL‐10, IL‐13, and INF‐γ) was measured in duplicate in collected supernatants by using Bioplex 200 System and commercial multiplexed kits according to the manufacturer's instructions (Bio‐Rad, Marnes‐la‐Coquette, France).
9
Histone, Antimicrobial, and Antibiotic Treatments
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Experiments involving histone treatments used calf thymus histone H2A (Sigma, St. Louis, MO), human histone H3 (Cayman Chemical, Ann Arbor, MI), or citrullinated human histone H3 (Cayman Chemical, Ann Arbor, MI). Experiments involving antimicrobial peptide treatment used the human cathelicidin LL-37 (Anaspec, Fremont, CA), FAM-LC-LL-37 (Anaspec, Fremont, CA), or magainin-2 (Anaspec, Fremont, CA). Experiments involving antibiotic treatments used kanamycin sulfate (Sigma), chloramphenicol (Sigma), or polymyxin B sulfate salt (Sigma).
10
Purification and Endotoxin Removal of Recombinant Toxoplasma Microneme Proteins
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The lactose-bound fraction (Lac+) was obtained from T. gondii as previously reported [12 (link),18 (link)]. Briefly, a preparation of soluble tachyzoite antigens (STAg) was loaded into a lactose-agarose column (Sigma-Aldrich, St. Louis, MO, USA) equilibrated with PBS containing 0.5 M NaCl. The material adsorbed to the resin was eluted with 0.1 M lactose in equilibrating buffer and dialyzed against ultrapure water. The lactose-bound fraction was denoted Lac+, and the presence of MIC1 and MIC4 in the fraction was confirmed. Cloning, expression, and refolding of the recombinant 6-histidine-tagged microneme proteins MIC1 and MIC4 were carried out as described previously [18 (link)]. All preparations were examined for endotoxin contamination using the Limulus Amebocyte Lysate Kit (QCL-1000; Lonza, Basel, Switzerland). The preparations of rMIC1 and rMIC4 contained 7.2 and 1.1 EU of endotoxin/µg protein, respectively. To further reduce the already low endotoxin concentrations, the preparations were applied to and eluted from polymyxin-B columns (Affi-Prep® Polymyxin Resin; Bio-Rad, Hercules, CA, USA). Prior to their use in cell-stimulation experiments, aliquots of the preparations were incubated with 50 µg/mL polymyxin B sulfate salt (Sigma-Aldrich) for 30 min at 37 °C to neutralize any residual endotoxin.
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