Monocytes activation assay

The procedure evaluated during our previous study was applied (Jarzembowski et al. 2018). As a reference, monocytes THP-1 cell line (TIB-202, ATCC, USA) was used. The cells were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100U/ml penicillin, 100 µg/ml streptomycin and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) (all from Sigma-Aldrich, Denmark).

Wells with biofilm were washed with 0,9% NaCl; suspension of monocytes (2,5*10⁵ of cells per well) was then added and incubated at 37 °C for 60 min on orbital shaker. Sterile wells were used as reference wells for adhesion and activation of monocytes.

For evaluation of monocytes’ activation, the number of monocytes and their morphology (described by FSC—forward scatter parameter featuring the size of the cell, and SSC—side scatter parameter referring to the observed cells’ granulation) were estimated by using the FACSVerse flow cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA) for predefined amount of time (time-restricted acquisition of data) and standardized with results obtained for reference wells.

The monocytes’ FSC and SSC parameters changes after the exposure to bacterial biofilm as compared with the monocytes exposed to reference well—empty, sterile well were considered factors reflecting the activation of the monocytes upon the contact with bacterial biofilm (FSC/K, SSC/K). The adherence of THP-1 cells to the bacterial biofilm was assessed by measuring the number of cells in the tube for predefined amount of time, collected from the biofilm coated well after 1 h incubation on orbital shaker in comparison to the number of cells in the tube collected from the reference well—without biofilm (ADH/K).