For assessing the primary response to stimulation, monocytes or macrophages were exposed for 24 h to LPS (positive control; from E. coli O55:B5 or K. pneumoniae; Sigma-Aldrich, Inc., St. Louis, MO, USA) or to increasing concentrations of rSmCD59.2, rSmTSP-2, unconjugated OMV, OMV:D, OMV:T, heat-killed K. pneumoniae (clinical isolates of both wild-type and carbapenemase-producing bacteria), or left untreated (medium/negative control).
For memory experiments, after the first exposure to stimuli for 24 h and supernatant collection, cells were washed and cultured with fresh culture medium for 6 additional days (one medium change after 3 days), to allow for the extinction of the activation induced by the previous stimulation. After this resting phase, the supernatant was collected, and cells were challenged for 24 h with fresh medium alone or containing a ten-fold higher concentration of stimuli. All supernatants (after the first stimulation, after the resting phase and after the challenge phase) were frozen at −20 °C for subsequent cytokine analysis. By visual inspection, cell viability and cell number did not substantially change in response to the different treatments.
For memory experiments, after the first exposure to stimuli for 24 h and supernatant collection, cells were washed and cultured with fresh culture medium for 6 additional days (one medium change after 3 days), to allow for the extinction of the activation induced by the previous stimulation. After this resting phase, the supernatant was collected, and cells were challenged for 24 h with fresh medium alone or containing a ten-fold higher concentration of stimuli. All supernatants (after the first stimulation, after the resting phase and after the challenge phase) were frozen at −20 °C for subsequent cytokine analysis. By visual inspection, cell viability and cell number did not substantially change in response to the different treatments.