Human/Mouse/Rat Total HSP70/HSPA1A DuoSet IC ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.	-When mixing or reconstituting protein solutions, always avoid foaming. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution.

Upstream tips
-Bring all reagents to room temperature before use. -Reconstitute the Human Cytochrome c Standard with deionized or distilled water. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.
Protocol tips
-When mixing or reconstituting protein solutions, always avoid foaming. -Keep Substrate solution protected from light. -Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution.

Publication protocol

BALB/c mice were injected intraperitoneally with 10 μg LPS (Sigma-Aldrich) in 1 ml sterile saline. Peritoneal macrophages were harvested 3 days post LPS injection by peritoneal lavage with 6 mL cold PBS and collected by centrifugation. Macrophages were enriched by adherence to plastic, recovered overnight and re-stimulated in vitro with 100 ng/mL LPS and 25 U IFN-γ (R&D systems) at 37°C or 39.5°C for indicated time points. Supernatant was collected for the presence of TNF-, IL-1 (Biolegend), IL-6 (BD Biosciences) and HSP70 (R&D systems) by ELISA. In some experiments, macrophages were treated with HSP70 inhibitors (KNK437, EMD chemicals; Pifithrin-μ, Sigma-Aldrich) together with LPS/IFN-γ.

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Manufacturer protocol

Download the product protocol from R&D Systems for Human/Mouse/Rat Total HSP70/HSPA1A DuoSet IC ELISA below.

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