Invitrogen elisa kit

The secretion of growth factors by hCE sheets into culture medium was evaluated by enzyme-linked immunosorbent assay (ELISA). Briefly, after 2 passages, human keratinocytes were cultured with 30 mL of conditioned medium in a T150-flask for 24 hours and the medium was then harvested and stored at −80°C.
After thawing, the samples were centrifuged at 18,800g for 10 minutes to remove cell debris and analyzed for the release of growth factors. Basic fibroblast growth factor (bFGF), platelet-derived growth factor-AA (PDGF-AA), TGF-α, TGF-β1, and keratinocyte growth factor (KGF) were measured using the Quantikine ELISA kit (R&D Systems, Minneapolis, Minn), and interleukin-1α (IL-1α), IL-1β, and vascular endothelial growth factor (VEGF) were measured using the Invitrogen ELISA kit (Invitrogen Corp., Camarillo, Calif) according to the manufacturers' instructions. The results were expressed as the amount of a growth factor released by 1 hCE sheet after 24-hour incubation compared with the fresh medium of the same composition as control.
Because irradiated 3T3 feeder cells used for keratinocyte culture may also release growth factors, confluent lethally irradiated 3T3 cells were incubated as above without human keratinocytes for 24 hours and their conditioned medium was analyzed by ELISA as described.

Following normal culture of THP-1 and TAMs for 48 h, cell culture supernatant was centrifuged for 20 min at 1,000 × g and at a temperature of 2–8°C. Cell debris and impurities were removed and supernatant was collected. Samples were stored at −20°C to avoid repeated freezing and thawing. These assays were performed according to the manufacturer's instructions, using the Invitrogen ELISA Kit (cat. no., KHC0101; Thermo Fisher Scientific, Inc.) for human IL-10. The standard curve assay range was 7.8–500 pg/ml, sensitivity <1 pg/ml.