Fig. 3

Heat shock response test in blood of SARS-CoV-2 patients. After harvesting, whole blood is immediately incubated at two different temperatures: 37 °C (control) and 42 °C (heat stressed) for 2 h in water bath (with gentle mix every 15 min). After the incubation, total blood is centrifuged to isolate plasma/serum and PBMC through density gradient separation. Then, plasma can be used for the direct analysis of extracellular HSP72 (eHSP72) while PBMC can be prepared for the measurement of intracellular (iHSP72). The PMBC must be washed and treated to ensure the absence of erythrocytes. PBMC are resuspended in RPMI 1640 medium (pH 7,4 supplemented with 2% NaHCO₃, 10% bovine calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin), seeded in a 24-well flat bottom plate (1 × 106 cells/well) and placed in an incubator for 6 h (37 °C in a 5% CO₂), in order to recover from the HS and reach the peak of HSP70 expression. Cells are then removed from the incubator, appropriated lysed and the total content of proteins prepared for western Blot analysis. The difference between concentration at 37 °C and 42 °C is used as HSR index