A chemoattractant solution was made by preparing 100 nM N-Formyl-Met-Leu-Phe (fMLP, Millipore Sigma) in 0.4% BSA/RPMI, supplemented, with 0.1% w/v FITC-dextran (Millipore Sigma). Immediately following cell alignment, 5 µL this fMLP solution was introduced into the source reservoir and a thin layer of silicone oil (Cat #378399, MilliporeSigma) was applied to cover the openings to minimize evaporation. For continuous cell tracking, the device was imaged with a Nikon Ti-2 microscope. The images were analyzed using the FIJI cell tracking plugin from ImageJ 29 . The cell paths obtained were later parsed and analyzed using custom Python scripts to visualize raw data. For end-point analysis, the microfluidic device was incubated in a 37⁰C humidified incubator for two hours. The device was subsequently imaged and cell migration distance was measured using ImageJ. No slip conditions were applied to obtain the initial profile of the gradient due to flow. The simulation assumed chemoattractant solution was introduced into the device at a flow rate of 1 µl/s for 5 seconds.
N formyl met leu phe fmlp
Manufactured by Merck Group
Sourced in United States
N-formyl-Met-Leu-Phe (fMLP) is a synthetic tripeptide compound used in laboratory research. It functions as a chemoattractant, stimulating the migration and activation of certain immune cells, such as neutrophils and macrophages.
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Lab products found in correlation
Fib 3, by Enzyme Research (3 mentions)
Cytochalasin b, by Merck Group (2 mentions)
Protein a sepharose, by Merck Group (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Ti2 microscope, by Nikon (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Silicone oil, by Merck Group (1 mentions)
Zm241385, by Bio-Techne (1 mentions)
Mab24, by BioLegend (1 mentions)
Cgs 21680, by Merck Group (1 mentions)
Dihydrorhodamine 123, by Merck Group (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cd16 bv786, by BD (1 mentions)
Cd63 pecy7, by BD (1 mentions)
Rpmi 1640 solution, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin glutamax p s, by Thermo Fisher Scientific (1 mentions)
Bacterial lipopolysaccharide lps escherichia coli 0111 b4, by Merck Group (1 mentions)
18 protocols using n formyl met leu phe fmlp
1
Chemotaxis Assay Using Microfluidic Platform
2
Neutrophil Adhesion Receptor Characterization
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Monoclonal antibodies for flow cytometric detection of high affinity β2-integrin (mAb24), L-Selectin (Dreg-55, Dreg-56), CD11b (M170), CD18 (1B4), CD66b (G10F5) CD11a (HI111), PSGL-1 (PL-2, KPL-1), CXCR1(8F1/CXCR1), and CXCR2 (5E8/CXCR2) along with antibodies that block CD11b function (Mac-1 blocking, M1/70), fixation buffer, and IL-8 were purchased from Biolegend (San Diego, CA). Recombinant human ICAM-1-IgG and E-Selectin-IgG produced as Fc chimeric constructs were purchased from R&D Systems (Minneapolis, MN). Adenosine A2A receptor agonist CGS-21680, N-formyl-Met-Leu-Phe (fMLP), and ROS indicator Dihydrorhodamine 123 were purchased from Millipore Sigma (Burlington, MA). Adenosine A2A receptor antagonist ZM 241385 was purchased from Tocris Bioscience (Minneapolis, MN). Feraheme (AMAG Pharmaceuticals, Waltham, MA) was purchased from the UC Davis Medical Center Pharmacy.
3
Phenotypic Analysis of Neutrophil Activation
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Human neutrophils were infected with Y. pestis KIM6+ or S. aureus as described above in wells of 96 well plates. Additionally, neutrophils were left uninfected/untreated or stimulated with Phorbol 12-myristate 13-acetate (PMA, 1 μM, Sigma, St. Louis, MO), E. coli lipopolysaccharide (LPS, 1 μg/ml, Sigma), or N-formyl-Met-Leu-Phe (fMLP, 10 μM, Sigma) where indicated as positive controls for neutrophil activation. Cells were harvested from wells at 15 min and 4 h post-infection and stained for flow cytometry with a cocktail of fluorescently labeled antibodies against the following markers: CD88-PerCP/Cy5.5 (BioLegend, San Diego, CA), CD66b-Alexa647, CD62L-PETR, CD16-BV786, CD63-PECy7, CD64-Alexa700, CD11b-Alexa488, and CD54-BV421 (all from BD Immunocytometry, San Jose, CA). The fixable viability dye efluor780 (ThermoFisher) was also included and analysis was limited to live, efluor780low/negative cells. After staining, cells were washed with 2% FBS in PBS and resuspended in 2% paraformaldehyde in PBS prior to analysis on a LSR-II flow cytometer (BD Immunocytometry). Data were analyzed using FlowJo 10.2 software.
4
Neutrophil Stimulation and Cytokine Release
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Purified neutrophils (5×106/mL, 500 μL) were incubated in RPMI-1640 solution (Gibco, Carlsbad, CA) supplemented with 5% fetal bovine serum (PAA Laboratories, Etobicoke, ON), 1% penicillin/streptomycin/GlutaMAX (P/S) (Gibco) and 25 mM HEPES (Sigma, Oakville, ON), and termed RPMI (for complete RPMI-1640 solution). Neutrophils were then stimulated for 2 and 24 hours with control vehicle (PBS), N-Formyl-Met-Leu-Phe (fMLP; 10-7 M) (Sigma, Oakville, ON), bacterial lipopolysaccharide (LPS; Escherichia coli 0111:B4; 1 μg/mL) (Sigma, Oakville, ON) or tumor necrosis factor-α (TNF-α; 10 ng/mL) (Peprotech, Rocky Hill, NJ) at 37°C, 5% CO2. Upon neutrophil stimulation, cells were centrifuged at 900 g for 7 minutes and supernatants stored at −80°C for future quantifications by ELISA of VEGF-A165, IL-1RA and IL-8 (R&D Systems). The selected aforementioned agonists (i.e. fMLP, LPS or TNF-α) were used because of their capacity to promote VEGF-A165, IL-1RA and IL-8 release by the neutrophils [28 (link)-31 (link)].
5
Boyden Chamber Assay for PMN Migration
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PMN migration was investigated by the Boyden chamber assay modified as previously described [46 (link)]. Briefly, after instrument assembly, PMN at 1 × 106 cells/mL in RPMI 1640 medium were placed in the upper chamber alone or together with the test substance, while the lower chamber contained medium alone (spontaneous migration) or added with 10 ng/mL interleukin (IL)-8 (Sigma–Aldrich, code: I1645) or 0.1 μM N-formyl-Met-Leu-Phe (fMLP, Sigma–Aldrich, code: F3506) (stimulated migration). Chambers were separated by a 3 μm pore-sized filter. After a 90-min incubation at 37 °C, the filter was harvested, dehydrated, fixed, and finally stained with haematoxylin. PMN migration was then quantified by light microscopy measuring the distance (in μm) from the surface of the filter to the leading front of cells.
6
Granulocyte Activation Marker Expression
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To study the expression of activation markers on granulocytes, 600 μl of heparinised venous blood is divided over 3 polystyrene tubes (200 μl/tube). After a pre-incubation of 5 minutes in a 37°C waterbath, a 5 minutes stimulation at 37°C is performed with N-Formyl-Met-Leu-Phe (FMLP, 10−5 M; Sigma, Saint Louis, MO, USA) or eotaxin (10−7 M; R&D systems, Abingdon, UK). Subsequently, 4 ml of FACS lysing solution (BD) is added and after an incubation period of 15 minutes at room temperature the red blood cells is lysed while white blood cells, including granulocytes, become fixed. Cells are washed with RPMI 1640 containing 10% heat-inactivated FCS and then resuspended in RPMI 1640 containing 10% of heat-inactivated foetal calf serum (FCS) and 10% dimethyl sulfoxyde (DMSO). Cryovials containing the cell suspension are placed at −80°C for minimum of 4 hours, followed by storage in liquid nitrogen until analysis.
7
Neutrophil Elastase Inhibition Assay
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Poloxamer 407 (BASF, the Netherlands), PBS (phosphate buffered saline, 10 mM phosphate, pH 7.4, 140 mM NaCl, Fresenius Kabi, the Netherlands), IMDM (Iscove's modified Dulbecco's medium, Lonza, Belgium), human elastase (EPC, Missouri, USA), cytochalasin B (Sigma, Germany), N-formyl-Met-Leu-Phe (fMLP; Sigma, Germany). PTG (PBS supplemented with 0.1% Tween 20 and 0.2% gelatin A), TMB (3,3′,5,5′ tetramethylbenzamidine, Uptima INTERCHIM, France), streptavidin horseradish peroxidase (HRP) (Amersham Life Science, UK), 96-well maxisorb plate (Nunc, Denmark), 96-well polysorb plate (Nunc, Denmark). Biacore buffer HBS-PE (GE Healthcare, the Netherlands). Ampex red (Molecular Probes/Thermo Fisher Scientific, US), HRP (Sigma, Germany).
8
Fibrinogen Fragment Interaction Assay
9
Intracellular Calcium Signaling Mechanisms
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N-formyl-met-leu-phe (fMLP), SKF96365, 2-Aminoethoxydiphenyl Borate (2-APB), thapsigargin (TG), and CaCl2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluo-4 AM was purchased from Invitrogen (Thermo Fisher Scientific Corporation, Waltham, MA, USA). Goat polyclonal anti-protein kinase B (Akt) antibody, rabbit polyclonal anti-phosphor-Akt, and anti-STIM1 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal anti-Src antibody and rabbit polyclonal anti-phosphor-Src antibody were purchased from Cell Signaling Technology (Boston, MA, USA). CSE was extracted from lit cigarettes, leached, dissolved in 2.5 mL of phosphate buffered solution, and then sterilized by filtration.
10
Fibronectin Extracellular Matrix Assay
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