Polymyxin b

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

Polymyxin B is a polypeptide antibiotic. It functions as an antimicrobial agent, specifically targeting Gram-negative bacteria.

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42 protocols using polymyxin b

Isolation and Culture of Human and Murine Macrophages

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Human blood samples from healthy volunteers were collected with heparin. Peripheral blood mononuclear cells were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Amersham). Cells were resuspended (10⁷ cells/mL) in RPMI-1640 with L-glutamine containing 10% heat-inactivated fetal bovine serum (FBS; Wisent) and seeded onto 18 mm glass coverslips (Fisher Scientific) at 5 x 10⁵ cells/coverslip. After 1 h at 37°C, non-adherent cells were removed by multiple washes. Adherent cells were incubated in RPMI-1640 with 10% FCS, 1 ng/mL human M-CSF and 100 U/mL penicillin, 100 μg/mL streptomycin and 10 μg/mL polymyxin B (Invitrogen) for 7–14 days.
To prepare bone marrow derived macrophages, femurs and tibias were dissected form euthanized mice. The marrow was extracted by perfusion of DMEM with a 21-gauge needle to produce a single-cell suspension, which was plated in DMEM with 10% FBS, 1 ng/mL murine M-CSF and 100 U/mL penicillin, 100 μg/mL streptomycin and 10 μg/mL polymyxin B (Invitrogen). After removal of non-adherent cells, macrophages were cultured for 7 days to allow complete maturation.

Jaumouillé V., Farkash Y., Jaqaman K., Das R., Lowell C.A, & Grinstein S. (2014). Actin cytoskeleton reorganization by the tyrosine kinase Syk regulates Fcγ receptor responsiveness by increasing its lateral mobility and clustering. Developmental cell, 29(5), 534-546.

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Mycobacterial Colony Enumeration

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Spleens and livers were homogenized in PBS with 0.5% Tween80 (Sigma-Aldrich) and serial dilutions were plated on 7H11 agar (Difco) supplemented with 10% OADC, 25 mg carbenicillin and 100,000 U polymyxin B (Gibco, Invitrogen, Auckland, New Zealand). Plates were incubated at 37°C and bacterial counts performed after 2–3 weeks growth.

Prendergast K.A., Daniels N.J., Petersen T.R., Hermans I.F, & Kirman J.R. (2018). Langerin+ CD8α+ Dendritic Cells Drive Early CD8+ T Cell Activation and IL-12 Production During Systemic Bacterial Infection. Frontiers in Immunology, 9, 953.

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PBMC-based IL-6 Response to SARS-CoV-2 Spike

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PBMC, 4x105 cells per well, were cultured in 48 wells plate in presence of RPMI 1640 (Gibco, 61870- 010) completed with 5% of heat inactivated AB human serum (Sigma, H4522). IL-6 secretion was assessed in PBMC culture supernatant 2, 15 and 24 h after recombinant Spike exposure, by ELISA using BD Opt EIA Set Human IL-6 (BD, 555 220) according to supplier’s recommendations. To analyze a possible contribution of endotoxin in the stimulation of IL-6 response, cells were treated during 24 h with a combination of 10 ng/mL LPS-EK (Invitrogen, Tlrl-eklps), 12.5 μg/mL Polymyxin B (inhibitor of endotoxin, Invitrogen, Tlrl-pmb), 2 μg/mL recombinant non-stabilized trimer Spike SARS-CoV-2 (ACROBiosystems, USA; SPN-C52H8) or its buffer in quantities equivalent to 2 μg/mL. IL-6 was measured (triplicates) on culture cell supernatants using the IL-6 ELISA assay kit BD Opt. EIA human IL-6 ELISA set (BD, 555 220) and GloMax plate reader (Promega, GM3000).

Charvet B., Brunel J., Pierquin J., Iampietro M., Decimo D., Queruel N., Lucas A., Encabo-Berzosa M.D., Arenaz I., Marmolejo T.P., Gonzalez A.I., Maldonado A.C., Mathieu C., Küry P., Flores-Rivera J., Torres-Ruiz F., Avila-Rios S., Salgado Montes de Oca G., Schoorlemmer J., Perron H, & Horvat B. (2023). SARS-CoV-2 awakens ancient retroviral genes and the expression of proinflammatory HERV-W envelope protein in COVID-19 patients. iScience, 26(5), 106604.

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Profiling T-cell Activation Responses

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T-cells purified as described in [13 (link)] were stimulated with S100A4 (1 μg/ml) for 19 h in presence of 10 μg/ml Polymyxin B (Invitrogen). Total RNA was isolated using a NucleoSpin® TriPrep kit (Macherey-Nagel). First-strand cDNA synthesis was performed using Super-Script III RT according to the manufacturer instructions. qRT-PCR was performed using a LightCycler 2.0 instrument (Roche Applied Science, USA). The expression level relative to the housekeeping GAPDH gene, as a control, was calculated. The PCR analysis was repeated 3 times using independent primary T-cell isolation. The primers used in this work are presented in Table 1.Table 1

List of primers used for analysis

Gene	Forward primer	Reverse primer
FN	5′-TGCCGCAACTACTGTGAT-3′	5′GAATCCTGGGCTGGAGTA--3′
G-CSF	5′-CAGATCACCCAGAATCCAT-3′	5′-CTCTCGTCCTGACCATAGTG-3′
Jak3	5′-TGGCCACTGAGGACTTCTCT-3′	5′-GGATGGCACTGGTCAAATCT-3′
IL6	5′-ACAAGAAAGACAAAGCCAGA-3′	5′-TAGCCACTCCTTCTGTGACT-3′
GATA3	5′-CTGGAGGAGGAACGCTAATG-3′	5′-GTTGAAGGAGCTGCTCTTGG-3′
IL10	5′-TCTCCCCTGTGAAAATAAGA-3′	5′-TCCAGCAGACTCAATACACA-3′
Tyk2	5′-ATCCGTTTGTACAGGCCAAG-3′	5′-GCTGTGTGATGGGGAACTTT-3′
CD40	5′-GGCTTCGGGTTAAGAAGGAG-3′	5′-GCAGGGATGACAGACGGTAT-3′
CTLA4	5′-GGATCCTTGTCGCAGTTAGC-3′	5′-AAACGGCCTTTCAGTTGATG-3′
TGFβ	5′-TGCGCTTGCAGAGATTAAAA-3′	5′-CGTCAAAAGACAGCCACTCA-3′
GAPDH	5′-TCATCCCTGCATCCACTG-3′	5′-TAGGAACACGGAAGGCCA-3′

Grum-Schwensen B., Klingelhöfer J., Beck M., Bonefeld C.M., Hamerlik P., Guldberg P., Grigorian M., Lukanidin E, & Ambartsumian N. (2015). S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance. BMC Cancer, 15, 44.

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Comprehensive Colon Cancer Cell Line Protocol

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Mouse colon carcinoma cell line CT26.CL25 (CRL-2639), human colon carcinoma cell lines SW620 (CCL-227), HT29 (HTB-38), COLO 205 (CCL-222) and Caco-2 (HTB-37), human neutrophilic promyeloblast cell line HL-60 (CCL-240), human monocyte/macrophage cell line THP-1 (TIB-202) and human lymphoblast cell line Jurkat (TIB-152) were purchased from American Type Culture Collection (ATCC, Manassas, VA). RPMI 1640 and Eagle's minimal essential medium (MEM) was obtained from Mediatech, Inc. (Manassas, VA). Macoy’s 5A modified medium was purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was from EQUITECH-BIO Inc. (Kerrville, TX). Penicillin-streptomycin stock was purchased from Lonza Rockland, Inc. (Allendale, NJ). Lipopolysaccharides from Escherichia coli O111:B4 was purchased from Sigma. Phycoerythrin (PE) conjugated antibodies targeting CD4 (H129.19), CD8b (YTS156.7.7), CD19 (6D5), dendritic cells (DCs) marker (33D1), LY6G (1A8), CD68 (FA-11) and mouse IgG1, κ isotype (MOPC-21) were purchased from BioLegend (San Diego, CA). OxPAPC (TLR2 and TLR4 inhibitor) and Polymyxin B (TLR4 inhibitor) were from InvitroGen (San Diego, CA).

Ishiguro S., Uppalapati D., Goldsmith Z., Robertson D., Hodge J., Holt H., Nakashima A., Turner K, & Tamura M. (2017). Exopolysaccharides extracted from Parachlorella kessleri inhibit colon carcinoma growth in mice via stimulation of host antitumor immune responses. PLoS ONE, 12(4), e0175064.

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Investigating Toll-like Receptor Signaling

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Cell culture media, fetal bovine serum (FBS), penicillin G, streptomycin, and β-mercaptoethanol were purchased from GIBCO. Gel-filtration chromatography -purified Escherichia coli O127:B8 LPS was purchased from Sigma. Antibodies (Abs) for total and phospho-specific MAPKs were purchased from Cell Signaling Technology. The MAPK inhibitors: SB203580 (p38) and SB202474 (the negative control for SB203580), U0126 (ERK), and U0124 (the negative control for U0126) and SP600125 (JNK) were purchased from Calbiochem. Polymyxin B, CLI-095, Pepinh-Control, Pepinh-MYD, Pepinh-TRIF, and Lipofectamine2000 were purchased from Invitrogen. Dual Luciferse Reporter Assay Kits and Cyto Tox 96 Non-Radioactive Cytotoxicity Assay Kits were purchased from Promega.

Bi L., Pian Y., Chen S., Ren Z., Liu P., Lv Q., Zheng Y., Zhang S., Hao H., Yuan Y, & Jiang Y. (2015). Toll-like receptor 4 confers inflammatory response to Suilysin. Frontiers in Microbiology, 6, 644.

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Cultivation and Biofilm Formation of Helicobacter pylori

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H. pylori Sydney strain 1 (SS1) (62 (link)) and all other H. pylori strains used in this study are listed in Table 1. Strains were grown on Columbia horse blood agar (CHBA) containing 0.2% β-cyclodextrin, 10 μg/ml vancomycin, 5 μg/ml of cefsulodin, 2.5 U/ml polymyxin B, 5 μg/ml trimethoprim, and 8 μg/ml amphotericin B (all chemicals from Thermo Fisher or Gold Biotech). Cultures were grown under microaerobic conditions (5% O₂ and 10% CO₂) at 37°C. For liquid culture and biofilm assay, H. pylori was grown in brucella broth (Difco) containing 10% heat-inactivated fetal bovine serum (FBS) (BB10 [Gibco/BRL]) with constant shaking under microaerobic conditions. For biofilm formation, several conditions were tested, including brucella broth containing different percentages of FBS (BB2, BB6, and BB10) and Ham’s F-12 (PAA Laboratories GmbH, Pasching, Austria) containing 10% or 2% FBS (HAMS10 and HAMS2, respectively).

Hathroubi S., Zerebinski J, & Ottemann K.M. (2018). Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella. mBio, 9(5), e01973-18.

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In Vitro Characterization of P. aeruginosa

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Mucoid (ATCC 39324) and nonmucoid (ATCC 27318) strains of P. aeruginosa were purchased from the American Type Culture Collection (Manassas, VA, USA). Cation-adjusted Müller–Hinton broth CA-MHB from BD (Franklin Lakes, NJ, USA) was used to grow P. aeruginosa for determination of in vitro antimicrobial activity and time-kill assays. Todd–Hewitt media supplemented with yeast extract was used for scanning electron microscopy (SEM), transmission electron microscopy (TEM), and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Gentamicin sulfate and polymyxin B were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Dosunmu E., Chaudhari A.A., Singh S.R., Dennis V.A, & Pillai S.R. (2015). Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect. International Journal of Nanomedicine, 10, 5025-5034.

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Synthesis of dansyl-polymyxin B conjugate

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Dansyl-Polymyxin B (dPM) was synthesized as described previously (47 (link)) with minor modifications. Briefly, Polymyxin B sulfate (Sigma-Aldrich) was dissolved in 0.1 M NaHCO₃ to reach a final concentration of 26 mM. Dansyl chloride (Acros Organics, Thermo Fisher Scientific, Waltham, MA) was dissolved in acetone to reach a final concentration of 46 mM. Polymyxin B was mixed with Dansyl chloride at a molecular ratio of 3:2, and the mixture was placed in the dark for 120 min at 37°C. After incubation, the dPM and the unreacted Dansyl chloride were separated by passage through a Sephadex G-25 column (Sigma-Aldrich). The fractions containing the dPM were extracted into n-butanol. The pure dPM was dissolved in the buffer of 5 mM HEPES (pH 7.0) and stored in aliquots at −20°C. The concentration of the dPM was determined by dinitrophenylation assay (48 (link)).

Lin Y., Sanson M.A., Vega L.A., Shah B., Regmi S., Cubria M.B, & Flores A.R. (2020). ExPortal and the LiaFSR Regulatory System Coordinate the Response to Cell Membrane Stress in Streptococcus pyogenes. mBio, 11(5), e01804-20.

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Antibiotic Susceptibility Testing Protocol

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The antibacterial susceptibility testing was performed usingthe disk diffusion and the VITEK 2 system (bioM’erieux, Marcy l’Etoile, France) and interpreted by the protocol specified in the guidelines of Clinical and Laboratory Standards Institute (CLSI, 2015). The antibacterial agents included cefoxitin, ceftriaxone, cefotaxime, cefepime, ceftazidime, cefazolin, moxifloxacin, ofloxacin, levofloxacin, gentamicin, amikacin, amoxycillin, minocycline, piperacillin, azithromycin, nitrofurantoin, polymyxin B, and meropenem (Thermo Fisher Scientific, USA). MDR isolates were defined as resistant to at least three different classes of antibiotics. Isolates were defined as extensively drug resistant (XDR) if they were not susceptible to at least one agent in all but two or fewer antibacterial categories (ie, bacterial isolates remained susceptible to only one or two antibacterial categories).

Qian W., Li X., Yang M., Liu C., Kong Y., Li Y., Wang T, & Zhang Q. (2022). Relationship Between Antibiotic Resistance, Biofilm Formation, and Biofilm-Specific Resistance in Escherichia coli Isolates from Ningbo, China. Infection and Drug Resistance, 15, 2865-2878.

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