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)].
Bacterial lipopolysaccharide lps escherichia coli 0111 b4
Manufactured by Merck Group
Bacterial lipopolysaccharide (LPS; Escherichia coli 0111:B4) is a complex glycolipid component of the outer membrane of Gram-negative bacteria. It is a well-characterized endotoxin that can trigger inflammatory responses in animals and humans.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 solution, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin glutamax p s, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by GE Healthcare (2 mentions)
Tumor necrosis factor α tnf α, by Thermo Fisher Scientific (1 mentions)
Vegf a165, by R&D Systems (1 mentions)
N formyl met leu phe fmlp, by Merck Group (1 mentions)
Il 1ra, by R&D Systems (1 mentions)
Tumour necrosis factor α tnf α, by Thermo Fisher Scientific (1 mentions)
2 protocols using bacterial lipopolysaccharide lps escherichia coli 0111 b4
1
Neutrophil Stimulation and Cytokine Release
2
Activation of Neutrophils by Inflammatory Stimuli
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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 named RPMI (for complete RPMI‐1640 solution). Neutrophils were then stimulated for 2 h with control vehicle (PBS), tumour necrosis factor‐α (TNF‐α; 10 ng/mL) (Peprotech, RockyHill, NJ) or bacterial lipopolysaccharide (LPS; Escherichia coli 0111:B4; 1 μg/mL) (Sigma) at 37°C, 5% CO2. Upon stimulation, neutrophils were centrifuged at 900 g for 7 min and supernatants stored at −80°C. The selected aforementioned agonists (LPS or TNF‐α) were used based on their corresponding capacity to promote VEGF, IL‐1RA, IL‐6, and IL‐8 release by the neutrophils.21 , 22
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