P62 elisa kit

Blood was collected from a peripheral vein preoperatively into a sterile heparin-containing tube and sent to the laboratory for analysis. The isolation of PBMCs was performed by Ficoll-Hypaque gradient centrifugation. The PBMC fraction was gently removed and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium for two cycles of centrifugation and resuspension. The nal suspension was adjusted to a concentration of 5x10 6 cells/ml. The PBMCs were centrifuged again and 130 µl of a cell lysate buffer (1% Triton X 100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 20 mg/mL deoxyribonuclease, 100 mmol/L protease inhibitor cocktail (Sigma, St Louis, Missouri) in 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, and 1 mmol/L ethylene glycol tetraacetic acid) was added to the pellets. The mixture was incubated for 30 min and the lysed PBMCs were centrifuged at 11000 rpm for 10 min. The supernatant was collected and concentrations of HSPA1A and p62 were quantitated using commercial ELISA kits (HSPA1A ELISA kit from R&D Systems, Minneapolis, MN, p62 ELISA kit from Enzo Life Sciences, Farmingdale, New York). The lower limit of detection was 156 pg/ml for HSPA1A and 100 pg/ml for p62. Values were converted to ng/ml by comparison to a standard curve generated in parallel to each assay.