Formyl met leu phe fmlp

Manufactured by Merck Group

Formyl-Met-Leu-Phe (fMLP) is a synthetic tripeptide compound commonly used in laboratory research. It is a chemoattractant, which means it has the ability to attract and activate specific cells, such as neutrophils and other immune cells. The core function of fMLP is to serve as a tool for studying cell signaling and chemotaxis in various in vitro and in vivo experimental models.

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N-formyl-Met-Leu-Phe (fMLP,

7 protocols using formyl met leu phe fmlp

Analyzing Neutrophil Properties

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Serially collected cells were selected using the Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), centrifuged onto glass slides using a Shandon Cytospin® 4 cytocentrifuge (Thermo Fisher Scientific), fixed immediately with PBS containing 4% paraformaldehyde, subjected to May–Giemsa and myeloperoxidase staining and immunostaining for lactoferrin, and analyzed by microscopy, as previously reported^{27 (link),29 (link)}.
Chemotaxis was determined using a modified Boyden chamber method, as previously reported^{20 (link)}. Briefly, 500 μl of the reaction medium (Hank’s Balanced Salt Solution containing 2.5% FCS) with or without 10 nM formyl-Met-Leu-Phe (fMLP; Sigma-Aldrich) was placed into each well of a 24-well plate, and the cell culture insert (3.0-mm pores; Becton Dickinson) was gently placed into each well to divide the well into upper and lower sections. Floating cells were suspended to the upper well at a concentration of 3.5 × 10⁴ cells per well, allowing the cells to migrate from the upper to the lower side of the membrane for 4 h at 37 °C. After incubation, cells in the lower chamber were collected and counted by flow cytometry. Dihydrorhodamine assay was performed as previously reported^{27 (link)}.

Hamabata T., Umeda K., Kouzuki K., Tanaka T., Daifu T., Nodomi S., Saida S., Kato I., Baba S., Hiramatsu H., Osawa M., Niwa A., Saito M.K., Kamikubo Y., Adachi S., Hashii Y., Shimada A., Watanabe H., Osafune K., Okita K., Nakahata T., Watanabe K., Takita J, & Heike T. (2020). Pluripotent stem cell model of Shwachman–Diamond syndrome reveals apoptotic predisposition of hemoangiogenic progenitors. Scientific Reports, 10, 14859.

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Transmigration Assay for Neutrophil Migration

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Transmigration assays were performed according to previously described protocols (McCormick et al., 1993 (link)). Briefly, T84 cells were seeded on collagen-coated inverted inserts (diameter 12 mm; pore size 3 μm) at a concentration of ~7.5 × 10⁵ cells per insert in 100 μl of DMEM-F12 medium. The inserts were then placed in a 12-well plate and cultured until the monolayer were confluent (10–14 days). Prior to the infection, the cells were washed and incubated for 30 min a 37°C with DMEM-F12. The cells were infected on the apical surface with ~1 × 10⁶ of EHEC wild type strain 86-24 or its isogenic lpf1 and lpf2 single or lpf1 lpf2 double mutants for 90 min at 37°C in 5% CO₂. Neutrophil chemoattractant formyl-Met-Leu-Phe (fMLP) (0.2 μM, Sigma) was added as positive control. After infection, monolayers were washed 2 × in HBSS and 20 μl of solution containing ~1 × 10⁷ PMN/ml was added to the basolateral side. After 4 h, neutrophils that migrated into the lower chamber were collected for quantification with a fluorometer.

Vergara A.F., Vidal R.M., Torres A.G, & Farfan M.J. (2015). Long polar fimbriae participates in the induction of neutrophils transepithelial migration across intestinal cells infected with enterohemorrhagic E. coli O157:H7. Frontiers in Cellular and Infection Microbiology, 4, 185.

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Synthesis and Characterization of Bioactive Compounds

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Platelet-activating factor (PAF; β-acetyl-γ-O-hexadecyl-L-α-phosphatidylcholine), formyl-met-leu-phe (fMLP), and Gue1654 (7-(methylthio)-2-[(2,2-diphenylacetyl)amino]benzo[1,2-d:4,3-d′]bisthiazole) were obtained from Sigma-Aldrich, whereas interkleukin-8 (IL-8) was purchased from R&D Systems. 5-Oxo-ETE³⁵ and LTB₄³⁶ were prepared by chemical synthesis as previously described. All reactions were carried out using dry solvents under argon atmosphere. High-resolution mass spectra were recorded on an AccuTOF mass spectrometer by positive ion ESI mode with DART as an ion source. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ using TMS as an internal standard on a BRUKER AMX 400 MHz spectrometer at rt. All compounds were analyzed by TLC, HRMS and NMR. Prior to biological assay, the purity of all final compounds was determined to be >95% by NMR and HPLC. HPLC conditions: Waters 2695 Alliance System with Waters Novapak C18 (150 × 3.9 mm) column and photodiode array detector (Waters Model 2996), gradient mobile phase between H₂O/MeCN/MeOH (56:22:22) to H₂O/MeCN/MeOH (16:42:42) over 30 min, both solvents contained 0.02% acetic acid, and a flow rate was 1 mL/min.

Gore V., Gravel S., Cossette C., Patel P., Chourey S., Ye Q., Rokach J, & Powell W.S. (2014). Inhibition of 5-oxo-6,8,11,14-eicosatetraenoic acid-induced Activation of Neutrophils and Eosinophils by Novel Indole OXE Receptor Antagonists. Journal of medicinal chemistry, 57(2), 364-377.

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Antibody-Based Protein Interaction Study

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Antibodies to the following antigens were acquired commercially: GST (2624, Cell Signaling), His (2366, Cell Signaling), HA (MMS-101R, Covance), RAB21 (R4405, Sigma), RAB5 (D-11, Santa Cruz), EEA1 (612006, BD), p-S234-RPH3A (Thermo Fisher, PA1-4693), RPH3A (Aviva System Biology, ARP59498_P050), and PIP5K1C90 ([Di Paolo et al., 2002 (link)] kindly provided by Dr. Pietro De Camilli). Protein A/G-PLUS-agarose beads were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Formyl-Met-Leu-Phe (fMLP) was purchased from Sigma (St. Louis, MO). The cDNAs for RAB21, and RPH3A, were acquired from Open Biosystems (Lafayette, CO).

Yuan Q., Ren C., Xu W., Petri B., Zhang J., Zhang Y., Kubes P., Wu D, & Tang W. (2017). PKN1 directs polarized RAB21 vesicle trafficking via phosphorylation of RPH3A and is important for neutrophil adhesion and ischemia-reperfusion injury. Cell reports, 19(12), 2586-2597.

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Siderophore-Mediated Bacterial Interactions

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Iron-free Ent (E. coli), pyoverdine (Pseudomonas fluorescens), ferrichrome (Ustilago sphaerogena), deferoxamine mesylate salt, 2,3 dihydroxybenzoic acid (2,3-DHBA), PMA, LPS (E. coli 0128: B12), Ca⁺² ionophore (A23187), DMSO, ferric chloride, PIPES, Histopaque®-1077 and 1119, RPMI, LB, Dextran, BSA, PFA, Saponin, formyl-Met-Leu-Phe (fMLP) and kanamycin were procured from Sigma (St Louis, MO). Leukotriene B4 (LTB₄) was from Cayman Chemical (Ann Arbor, MI). Carrier-free mouse recombinant Lcn2 (free from endotoxin, siderophore, and iron) was obtained from Cell Signaling (Danvers, MA). Chrome azurol S (CAS) was purchased from Acros Organics (Geel, Belgium).

Saha P., Yeoh B.S., Olvera R.A., Xiao X., Singh V., Awasthi D., Subramanian B.C., Chen Q., Dikshit M., Wang Y., Parent C.A, & Vijay-Kumar M. (2017). Bacterial Siderophores Hijack Neutrophil Functions. Journal of immunology (Baltimore, Md. : 1950), 198(11), 4293-4303.

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Isolation and Stimulation of Neutrophils and Mast Cells

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Peripheral whole blood was obtained from consenting healthy adult donors in accordance with protocols approved by the University of Wisconsin-Madison Institutional Review Board. PMNs and MCs were isolated as previously described using a gradient method.^{24 (link)} A small percentage (<1%) of basophils and eosinophils are possible in the PMN isolation. The MC isolation procedure routinely results in approximately 80–90% MC purity with lymphocytes as the primary impurities. PMNs were adjusted to 1 × 10⁶ cells/ml in RPMI-1640 medium supplemented with 10% autologous human serum (AHS). The PMN suspension was split into two groups where one group received 100 nM of the bacterial peptide formyl-Met-Leu-Phe (fMLP; Sigma-Aldrich, St. Louis, MO). This concentration of fMLP is used to stimulate PMN degranulation.²⁵ PMNs were statically seeded onto PEG hydrogel, PDMS, TCPS and GP hydrogel surfaces in 48-well plates (0.5 ml/well for a density of approximately 5.3 × 10³ PMNs/mm²) and were incubated in a humidified atmosphere at 37°C with 5% CO₂ for between 2 and 24 hours. MCs were adjusted to 1.7 × 10⁶ cells/ml in RPMI-1640 medium supplemented with 10% AHS and were seeded as described below for the MC chemotaxis assay.

Cohen H.C., Lieberthal T.J, & Kao W.J. (2014). Poly(ethylene glycol)-containing hydrogels promote the release of primary granules from human blood-derived polymorphonuclear leukocytes. Journal of biomedical materials research. Part A, 102(12), 4252-4261.

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Neutrophil-like cell activation and manipulation

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Neutrophil-like dHL60 cells were activated with chemoattractant formyl-Met-Leu-Phe (fMLP; Sigma) at effective final concentration of either 25 nM (for imaging based assays) or 100 nM (for cellular biochemistry). Acute increase in membrane tension and stretching was done by adding equal volume of hypotonic buffer (H2O + 1 mM MgCl2 + 1.2 mM CaCl2) as described earlier (Graziano et al., 2019; (link)Houk et al., 2012) (link). mTOR kinase activity was inhibited by treating cells with 10 M KU-0063794 (Selleckchem) for 30-45 mins in plain RPMI-1640. PI3Kinase activity was inhibited by incubating cells with 1 M PIK-90 for 30-45 mins in plain RPMI as described earlier (Van Keymeulen et al., 2006) (link). ROCK inhibition was done by treating cells with 20 M Y-27632 for 20 min in plain RPMI prior to the imaging assay (Graziano et al., 2019) (link).

Saha S., Town J.P., & Weiner O.D. (2022). mTORC2 coordinates the leading and trailing edge cytoskeletal programs during neutrophil migration.

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