ELISA was used to measure cytokines as described previously [7 (link)]. For measuring cytokines in culture supernatants, the endometrial epithelial cells or bone marrow-derived cells were grown in 24 well plates at the cell density of 1.5 × 105/ml for MS74 [an immortalized human vaginal epithelial cell line kindly provided by Dr. Robert Schenken from Department of Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio; ref: [16 (link)]] or 1 × 106/ml for bone marrow-derived neutrophils. Cells were stimulated with LPS (Cat# tlrl-3pelps), Pgp3, LL-37 or Pgp3/LL-37 complex at different concentrations as indicated in individual experiments for 24h. The supernatants with or without dilutions were used for cytokine measurement. The LL-37/Pgp3 complex was formed by pre-incubating the two together in cell culture medium for 30min at 37°C. ELISA kits were used for measuring mouse IL-6 (Cat# 431301, Biolegend, San Diego, CA), mouse MIP-2 (Cat# DY452, R&D systems, Minneapolis, MN), human IL-6 (Cat# 555220) and human IL-8 (Cat# 555244, both from BD Bioscience, San Jose, CA) respectively.
Human il 8
Manufactured by BD
Sourced in France, Germany, United States
Human IL-8 is a cytokine that functions as a chemoattractant for neutrophils. It is a key mediator of the inflammatory response.
Automatically generated - may contain errors
5 protocols using human il 8
1
Cytokine Profiling in Immune Cell Cultures
2
Cytokine and hs-CRP Quantification in Patient Serum
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For analyses of IL-1 β, IL-6, IL-8, and TNF-α, the blood samples were collected from the patients at 8 am after an overnight fast. The serum samples were immediately centrifuged and were stored at -80°C until further determination. IL-1β, IL-6, and IL-8 were determined with the commercial enzyme-linked immunosorbent assay kits (human IL-1β: R&D Systems INC., Minnesota, USA; human IL-6: high sensitivity enzyme-linked immunosorbent assay, Immunotech S. A, Marseille, France; human IL-8: enzyme-linked immunosorbent assay; BD Bioscience, La Jolla, CA) as previously described [17 (link), 18 (link)]. The serum samples were stored in -80°C until further determination, and the serum levels of TNF-α were determined by a high sensitivity enzyme-linked immunosorbent assay kit (Biosource, International, Inc.) according to the manufacturer's instructions [19 (link)]. The serum levels of high sensitive C reaction protein (hs-CRP) were analyzed with a commercially available assay kit (hs-CRP test kits, Millipore Corp, Billerica, MA, USA), and the levels of hs-CRP were expressed as mg/L.
3
Murine and Human IL-22, IL-8 Secretion
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Murine and human IL-22 (R&D-Systems, Wiesbaden, Germany) and human IL-8 (BD Biosciences) secretion were determined by ELISA according to the manufacturer’s instructions.
4
Quantification of Immune Markers
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Immunoglobulin and cytokine levels in the serum or culture supernatant were measured using commercially available ELISA kits for human IL-6 (BD Biosciences, USA), human IL-8 (BD Biosciences), IgA Mouse Uncoated ELISA Kit with Plates (Thermo Fisher Scientific), and IgG (Total) Mouse Uncoated ELISA Kit with Plates (Thermo Fisher Scientific), according to the manufacturer’s instructions.
5
Quantifying ACE2 and Cytokine Levels
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ACE2 and ILs levels were measured by commercially available sandwich enzyme-linked immunosorbent assays (ELISAs) following manufacturer’s instructions. The following ELISA kits were employed: human IL-6 (BD Biosciences cat. 555220); human IL-8 (BD Biosciences cat. 555224); human IL-1β (BD Biosciences cat. 557953); human IL-10 (BD Biosciences cat. 555157), and human ACE2 duoset kit (R&D SYSTEMS cat. DY933–05).
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