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N formylmethionine leucyl phenylalanine fmlp

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N-formylmethionine-leucyl-phenylalanine (fMLP) is a synthetic peptide that is commonly used as a laboratory reagent. It is a chemoattractant that can stimulate the migration of certain immune cells, such as neutrophils, towards its source. fMLP is often used in in vitro experiments to study cellular signaling and chemotaxis.

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Lab products found in correlation

Amplex red kit, by Thermo Fisher Scientific (2 mentions) Gm csf, by Merck Group (1 mentions) G csf, by Merck Group (1 mentions) Tnf α, by Merck Group (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Cytospin 4, by Thermo Fisher Scientific (1 mentions) Macsxpress neutrophil isolation kit, by Miltenyi Biotec (1 mentions) Percoll gradient, by GE Healthcare (1 mentions) Infinite f200 pro plate reader, by Tecan (1 mentions) Amplex red, by Tecan (1 mentions) Amplex red, by Merck Group (1 mentions) L 15 media, by Thermo Fisher Scientific (1 mentions) Fibronectin, by Merck Group (1 mentions) Volocity software, by PerkinElmer (1 mentions) Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions) Genios plate reader, by Tecan (1 mentions) Unopsonized zymosan, by MP Biomedicals (1 mentions) Zymosan, by MP Biomedicals (1 mentions) Flow cytometry, by Beckman Coulter (1 mentions)

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Phenylalanine (126 citations) N-formylmethionine-leucyl-phenylalanine (3 citations)

8 protocols using n formylmethionine leucyl phenylalanine fmlp

1

Neutrophil Priming and ROS Measurement

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GM-CSF, G-CSF, TNF-α, and N-formylmethionine-leucyl-phenylalanine (fMLP) were purchased from Sigma-Aldrich (Milan, Italy). Neutrophils (3 × 106/mL) were incubated either with G-CSF or GM-CSF or primed with CSFs for 50 min and then stimulated with 1 μM fMLP. For ROS measurement experiments, DMSO was used as a negative control for fMLP and TNF-α (50 ng/ml) was used as positive control for priming. In some experiments, both CSFs were present together at low concentrations (0.005–0.1 ng/ml) both as direct stimulators or priming agents for fMLP.
Castellani S., D’Oria S., Diana A., Polizzi A.M., Di Gioia S., Mariggiò M.A., Guerra L., Favia M., Vinella A., Leonetti G., De Venuto D., Gallo C., Montemurro P, & Conese M. (2019). G-CSF and GM-CSF Modify Neutrophil Functions at Concentrations found in Cystic Fibrosis. Scientific Reports, 9, 12937.
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2

Neutrophil Isolation and Adhesion

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Live human neutrophils were isolated via negative magnetic antibody-based selection with the MACSxpress neutrophil isolation kit (Miltenyi Biotec) and resuspended in RPMI media with L-glutamine and HEPES (Life Technologies). In order to induce and maintain a normal cell spreading22 (link) and adhesion to microscope slide surface, quartz slides were coated with a 1 nM solution of N-Formylmethionine-leucyl-phenylalanine (fMLP, Sigma-Aldrich) for 60 minutes, then rinsed with distilled water and PBS. The cell suspension was pipetted onto an imaging chamber placed on the coated slide and sealed with a quartz coverslip. In order to induce additional spreading on the neutrophils, cells were prepared on fMLP coated quartz slides using a Cytospin 4 (Thermo-scientific) at 500 g for 5 minutes.
Ojaghi A., Fay M.E., Lam W.A, & Robles F.E. (2018). Ultraviolet Hyperspectral Interferometric Microscopy. Scientific Reports, 8, 9913.
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3

Neutrophil Migration Assay with IFNλ

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Neutrophils were isolated from blood samples over a 62.5% Percoll gradient (GE Healthcare) in Ca2+- and Mg2+-free HBSS as previously described (Mocsai et al, 2000 (link)) with a purity of more than 90%. 5 × 105 neutrophils were placed into a 24-well PET Transwell (8 μm pore membrane). As positive control of 100 nM N-formylmethionine-leucyl-phenylalanine (fMLP; Sigma-Aldrich) was added to the medium placed on the bottom of the wells. IFNλ1 or IFNλ4 were used as stimulus alone or in combination with fMLP. Neutrophils were allowed to migrate for 4 h. After 4 h, the medium was collected, and migrated neutrophils were counted using FACS. Data are reported of percentage of migrated neutrophils over the total number of neutrophils.
Coto-Llerena M., Lepore M., Spagnuolo J., Di Blasi D., Calabrese D., Suslov A., Bantug G., Duong F.H., Terracciano L.M., De Libero G, & Heim M.H. (2020). Interferon lambda 4 can directly activate human CD19+ B cells and CD8+ T cells. Life Science Alliance, 4(1), e201900612.
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4

NADPH-Oxidase Activity in Neutrophils

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Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity was assessed by measuring the release of hydrogen peroxide (H2O2) with an Amplex Red kit (Molecular Probes). Neutrophils (0.25 × 106) were left unstimulated in HEPES buffer or were stimulated for 30 minutes at 37°C with E. coli (OD625 = 0.2, strain ML-35), unopsonized zymosan (1 mg/mL), serum-treated zymosan (STZ, 1 mg/mL), phorbol 12-myrisatate 13-acetate (PMA, 100 ng/mL; Sigma Aldrich) or platelet-activation factor (PAF; 1 µmol/L; Sigma Aldrich)/N-formylmethionine-leucyl-phenylalanine (fMLP, 1 µmol/L; Sigma Aldrich) in the presence of Amplex Red (0.5 µmol/L) and horseradish peroxidase (1 U/mL). Fluorescence derived from Amplex Red conversion into Resorufin was measured at 30-second intervals for 30 minutes with an Infinite F200 PRO plate reader (Tecan). The activity of the NADPH oxidase of neutrophils was determined as nmol H2O2/min × 106 cells.
Martinez-Sanz P., Laurent A.R., Slot E., Hoogenboezem M., Bąbała N., van Bruggen R., Rongvaux A., Flavell R.A., Tytgat G.A., Franke K., Matlung H.L., Kuijpers T.W., Amsen D, & Karrich J.J. (2023). Humanized MISTRG as a preclinical in vivo model to study human neutrophil-mediated immune processes. Frontiers in Immunology, 14, 1105103.
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5

Neutrophil Motility and Uropod Formation Assay

Cited 1 time
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WT and TgSOD3 PMNs were harvested from saline- or LPS-injected mice
(SAL-PMN, LPS-PMN) and cultured in L-15 media (Thermo Fisher Scientific)
supplemented with 4.5 g/L glucose on glass chamber slides coated with 5
μg/ml fibronectin (Sigma). PMNs were stimulated with 1 μM in PBS
of the chemoattractant N-formylmethionineleucyl-phenylalanine (fMLP; Sigma) to
induce motility40 (link),41 (link) and allowed to settle for 12 minutes
before imaging. Images were acquired every 15 seconds for 20 minutes, and both
velocity and directional index for migrating cells were analyzed with Volocity
software (PerkinElmer, Waltham, MA). A directional index of 1 indicated movement
in a straight line while an index of 0 indicated no displacement from the
original position. All steps for migration assays were performed at 37°C.
Bright-field live cell movies were acquired at a frame rate of 12 images/minute
for a total of 24 hours to assess for PMN uropod formation in co-cultures. PMNs
were identified by their spheroid shape and high contrast ratio, and individual
cells were monitored for the extension of one or more uropods during the entire
acquisition period. Data were gathered from mixed cultures of OGD-exposed
cortical neurons (4 hours) and SAL- or LPS-stimulated PMNs harvested from WT and
TgSOD3 mice.
Mai N., Prifti V., Lim K., O’Reilly M.A., Kim M, & Halterman M.W. (2020). Lung SOD3 limits neurovascular reperfusion injury and systemic immune activation following transient global cerebral ischemia.. Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association, 29(9), 104942.
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6

Neutrophil Response to Macrophage Secretome

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Freshly isolated neutrophils were seeded at a density of 2.5 × 105 in each well of a 48 well plate. Cells were treated with 20% conditioned media, under 4 different conditions as follows: cRPMI (RPMI + 10% human serum), MΦ-CM from unstimulated hMDMs (UsCoM), MΦ-CM from Mtb-stimulated hMDMs (MtbCoM), or MΦ-CM from LPS-stimulated hMDMs (LPSCoM). Following one hour of incubation, the conditioned media was washed off and the cells were resuspended in fresh cRPMI supplemented with 1% FBS. Neutrophils were artificially activated using 1.25 ng/mL N-Formylmethionine-leucyl-phenylalanine (fMLP; Sigma-Aldrich, St. Louis, MO, USA), where applicable.
Murphy D.M., Walsh A., Stein L., Petrasca A., Cox D.J., Brown K., Duffin E., Jameson G., Connolly S.A., O’Connell F., O’Sullivan J., Basdeo S.A., Keane J, & Phelan J.J. (2024). Human Macrophages Activate Bystander Neutrophils’ Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis. International Journal of Molecular Sciences, 25(5), 2898.
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7

Measuring Hydrogen Peroxide Release

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The release of hydrogen peroxide was measured by using the Amplex Red kit (Molecular probes), as described earlier (19) . Note that 0.25 Â 10 6 cells were stimulated with Escherichia coli (OD625 ¼ 0.2, strain ML-35; 0.25 Â 10 9 per mL) in the presence of 2 mmol/L azide, platelet-activation factor (PAF; 100 nmol/L; Sigma-Aldrich)/Nformylmethionine-leucyl-phenylalanine (fMLP; 30 nmol/L; Sigma Aldrich), phorbol 12-myristate 13-acetate (PMA; 100 ng/mL; Sigma Aldrich), serum-treated zymosan (21) , or unopsonized zymosan (1 mg/mL; MP Biomedicals) for 30 minutes at 37 C. Every 30 seconds, fluorescence was assessed with a Genios plate reader (Tecan). Maximal slope of H 2 O 2 release was measured at 2-minute intervals at an excitation wavelength of 535 nm and an emission wavelength of 595 nm. Analysis is shown as the maximal slope in relative fluorescence units/minute.
Bouti P., Zhao X.W., Verkuijlen P.J.J.H., Tool A.T.J., van Houdt M., Köker N., Köker M.Y., Keskin O., Akbayram S., van Bruggen R., Kuijpers T.W., Matlung H.L, & van den Berg T.K. (2021). Kindlin3-Dependent CD11b/CD18-Integrin Activation Is Required for Potentiation of Neutrophil Cytotoxicity by CD47-SIRPα Checkpoint Disruption. Cancer immunology research, 9(2).
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8

Chemotaxis Assay for Immune Cells

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Migration of both untreated and LPS treated (24 hours) cells was analyzed using 24 well transwell plates (polycarbonate membrane with 5 μm pore, Corning, Corning, NY, USA). 105 cells in 100 μl RPMI-1640 medium were plated in the upper chamber, and the lower chamber was filled with 600 μl of chemoattractant solution or medium (negative control). For the untreated cells 62.5 nM N-Formylmethionine-leucyl-phenylalanine (FMLP, Sigma-Aldrich) was used as a chemoattractant and the migration was stopped after 1 hour. For the LPS treated cells we used 200 ng/ml CCL19 (BioLegend), and the migration was stopped after 2 hours for MDDCs and 4 hours for MDMs. At the end of the incubation time, EDTA was pipetted to the lower chamber without removing the membrane, at a final concentration of 12.5 mM. After 5 minutes at 37°C cells were resuspended from the lower chamber and the bottom of the membrane, placed on ice and counted immediately. The number of transmigrated cells was determined in a volume of 250 μl/sample by flow cytometry (Cytoflex, BC).
Lukácsi S., Gerecsei T., Balázs K., Francz B., Szabó B., Erdei A, & Bajtay Z. (2020). The differential role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in the adherence, migration and podosome formation of human macrophages and dendritic cells under inflammatory conditions. PLoS ONE, 15(5), e0232432.
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