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)].
Penicillin streptomycin glutamax p s
Manufactured by Thermo Fisher Scientific
Penicillin/streptomycin/GlutaMAX (P/S) is a commonly used cell culture supplement. It is a mixture of the antibiotics penicillin and streptomycin, along with the amino acid derivative GlutaMAX. This product is designed to provide antimicrobial protection and support cell growth in cell culture applications.
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Lab products found in correlation
Rpmi 1640 solution, by Thermo Fisher Scientific (2 mentions)
Bacterial lipopolysaccharide lps escherichia coli 0111 b4, by Merck Group (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 penicillin streptomycin glutamax p s
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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