Peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood samples by differential centrifugation using histopaque 1.077 (Sigma-Aldrich). Cells were washed, counted and seeded at 5 × 106 cells/mL in RPMI 1640 media supplemented with 2 mM L-glutamine, 10% heat-inactivated foetal calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin. PBMC were then stimulated with either 1 µg/mL LPS (Escherichia coli O55:B5; Sigma-Aldrich) or mock treated with PBS as controls. The PBMC were then cultured for 24 hours at 37 °C and 5% CO2, before the supernatant was harvested and stored at −20 °C. Cytokine concentrations were then measured by ELISA using commercial antibody pairs according to the manufacturer’s instructions (IL-10 and TNFα – ThermoFisher; IL-6, IL-8, IL-1β – Duosets, R and D Systems).
Il 10 and tnfα
Manufactured by Thermo Fisher Scientific
IL-10 and TNFα are cytokine proteins that play crucial roles in immune system regulation. IL-10 is an anti-inflammatory cytokine, while TNFα is a pro-inflammatory cytokine. These proteins are often measured in research and clinical settings to understand immune system function and response.
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2 protocols using il 10 and tnfα
1
Cytokine production of activated PBMC
2
Cytokine profiling of stimulated PBMCs
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The haematology profile was analysed on an Advia 2120 Hematology System (Siemens Healthcare Diagnostics), and the serum analyses for biochemistry were assayed using an Advia 1800 Chemistry System (Siemens Healthcare Diagnostics). For cytokine concentration, the peripheral blood mononuclear cells were obtained from heparinised blood samples by differential centrifugation using histopaque 1•077 (Sigma-Aldrich). Cells were washed, counted and seeded at 5 × 10 6 cells/ml in RPMI 1640 media supplemented with 2 mM L-glutamine, 10 % heatinactivated fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The peripheral blood mononuclear cells were then stimulated with either 1 μg/ml LPS (Escherichia coli O55:B5; Sigma-Aldrich) or mock-treated with PBS as controls. The peripheral blood mononuclear cells were then cultured for 24 h at 37°C and 5 % CO 2 , before the supernatant was harvested and stored at -20°C. Cytokine concentrations were then measured by ELISA using commercial antibody pairs according to the manufacturer's instructions (IL-10 and TNFα -ThermoFisher; IL-6, and IL-1β -R and D Systems).
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