Hsp70 high sensitivity elisa kit

Levels of Hsp70 in the serum and hippocampus were determined by enzyme‐linked immunosorbent assay (ELISA). The Hsp70 ELISA Kit (ab133060, Abcam) was used for assessing Hsp70 levels in the hippocampus. The Hsp70 High Sensitivity ELISA Kit (ab133061, Abcam) was used for assessing Hsp70 levels in mouse serum. The following kits were used for the detection of inflammatory cytokines in the hippocampus: Mouse IL‐6 ELISA kit (ab100713, Abcam) for IL‐6, Mouse IL‐1 beta ELISA Kit (ab197742) for IL‐1ß, and Mouse monocyte chemoattractant protein‐1 (MCP1) ELISA Kit (ab100722) for MCP1. ELISA was performed according to the manufacturer's instruction.

The tumor interstitial fluid was obtained by digesting tumors as described above. After removing cells by centrifugation, the supernatant was further purified by centrifugation at maximum speed for 10 min and stored at −70 °C until use. The protein concentration of the tumor effusate was measured using Bradford reagent (Thermo Fisher Scientific). In the subsequent measurements, an equal amount of protein was used. The hsp70 concentration was measured using a hsp70 High-Sensitivity ELISA kit (Abcam). The HMGB1 concentration was determined using a specific ELISA kit from Aviva Systems Biology (San Diego, CA, USA) in accordance with the manufacturer’s protocol. Absorbance was measured at 450 nm with PowerWave microplate spectrophotometer (BioTek, Winooski, VT). ATP content was evaluated using a luciferase-based ATP determination kit (Thermo Fischer Scientific) following the manufacturer’s instructions. Luminescence was measured with a Varioskan Flash microplate reader (Thermo Fischer Scientific).