Cytochalisin d

The human promyelocytic cell line HL-60 (ATCC CCL-240) was maintained in RPMI 1640 medium, GlutaMAX supplement (ThermoFisher) supplemented with 10% fetal bovine serum (FBS, ThermoFisher) at 37°C and 5% CO2 between 2.0 × 10⁵ and 1 × 10⁶ cells/ml. 4 × 10⁵ cells/ml were differentiated with 100 mM with N, N-dimethylformamide (DMF, Sigma). HL-60 cells are differentiated into granulocytes by 4–5 days of treatment and are ready to be used as phagocytes with a typical yield of 8-12 × 10⁵ cells/ml. For all host-pathogen interactions studies, RPMI 1640 Medium, no phenol red (ThermoFisher) with 2% of FBS was used. When desired, differentiated cells were stimulated for ROS production by using 10 μg/ml of phorbol 1-myristate 13-acetate (PMA, Sigma). When stated, 10 μg/mL cytochalisin D (Sigma) was used to inhibit phagocytosis. Permeabilization buffer containing PBS +0.2% saponin with or without 2% BSA was used for phagocytosis, NET, autophagic studies and bacterial survival evaluation.

PMA-stimulated dHL-60 (1 × 10⁶) were treated with 10 μg/mL cytochalisin D (Sigma) (for bacterial adhesion) or left untreated (total bacteria-dHL-60 interaction). They were infected with 5 × 10⁷ CFU (MOI 50) of M. catarrhalis or NTHi bacterial cells for 20, 45 and 75 min in RPMI 1640 (no phenol red) + 2% FBS medium and incubated at 37°C in the presence of 5% CO₂. After incubation, samples were fixed with 4% (v/v) formaldehyde (Sigma) for 1 h and M. catarrhalis and NTHi bacteria were detected by staining for UspA2 or whole cell, respectively (rabbit polyclonal antibodies diluted 1:1000 in permeabilization buffer). Samples were incubated for 20–30min at RT in the dark with 100 μL of permeabilization buffer containing a secondary rabbit anti-mouse immunoglobulin G (whole molecule) Alexa fluor 633-conjugated (Invitrogen) diluted 1:500. After two washes with PBS, dHL-60 were evaluated by flow cytometry and expressed as the percentage of Alexa Fluor 647 positive cells (untreated samples) or the percentage of cells with bacteria attached (cytochalisin D). The percentage of phagocytosis was calculated subtracting the percentage of adhesion to the total interaction. All data were collected using a BD FACS CANTO II (BD Bioscience) by acquiring 100,000 events, and the data analysed using the Flow-Jo software (v.8.6, TreeStar Inc).