Lionheart fx automated microscopy

THP-1 cells were treated with a final concentration of 50 nM Phorbol 12-Myristate 13-Acetate (PMA; Sigma Aldrich, St. Louis, MO, USA) for 48 h. Infection with M. abscessus harboring pTEC15 green fluorescent or luciferase was carried out at 37 °C in the presence of 5% CO₂ for three hours at a multiplicity of resistance (MOI) of 2:1. After extensive washing with PBS, cells were incubated containing 50 ug/mL gentamycin for 30 min and washed again with PBS. Then 200 uL RPMI media containing DMSO (negative control) and 200 uL RPMI media containing the indicated concentration of CC were incubated for 3 to 4 days. The macrophages were stained with SYTO 60 (Invitrogen, Eugene, OR, USA) dye at a final concentration of 5 µM for 30 min at 37 °C, 5% CO₂. Luminescence was measured on day four by using a Synergy H1 microplate reader (Bio-Tek, Winooski, VT, USA). Fluorescent images of live cells were captured by automated microscopy using a Lionheart™ FX automated microscopy (Bio-Tek, Winooski, VT, USA). The Gen5^TM 3.05 software object feature enables the identification of cells within the imaging field.

THP-1 cells were treated with a final concentration of 50 nM PMA (Sigma Aldrich, St. Louis, MO, USA) for two days. Infection with M. abscessus harboring green fluorescent (pTEC15) or luciferase was carried out at 37 °C for three hours at an MOI of 2:1. After three washes with PBS, cells were incubated containing 50 μg/L gentamycin for 30 min and washed again. Then, 200 uL RPMI 1640 media containing the indicated concentration of 10-DEBC was left for four days. The macrophages were stained with SYTO 60 (Invitrogen, Waltham, MA, USA) dye at a final concentration of 5 µM for 30 min at 37 °C. Luminescence was measured on day four using a Synergy H1 microplate reader (Bio-Tek, Winooski, VT, USA).
iMAC cells were previously described and provided kindly by Dr. Jung-Hyun Kim [21 (link)]. The iMAC cell’s infection method using M. abscessus harboring green fluorescent was the same as differentiated THP-1 cells. Live-cell images were captured by automated microscopy using Lionheart™ FX automated microscopy (Bio-Tek, Winooski, VT, USA). The Agilent BioTek Gen5^TM 3.05 software object feature enables the calculation of cells within the imaging field.

Immunofluorescence (IF) was performed as follows. Coverslips coated with poly-D-lysine (Gibco, USA) were incubated for 30 min at 37°C. Raw264.7 cells were seeded at a density of 1 × 10⁵ cells per slide and incubated for 24 h. The cells were treated with CH for 24 h, fixed at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, and washed with PBS 4 times. The cells were incubated for 30 min with a blocking solution (0.5% Ez-Block Chemi) and incubated overnight at 4°C for 24 h with antibodies against PB1, p-IRF3, and MAVS. The cells were washed with PBS, incubated for 30 min with secondary antibody and lysotracker, washed with PBS, and stained with Hoechst 33342 for 5 min. The coverslips were mounted onto slides with mounting medium and visualized via fluorescence microscopy (Lionheart FX automated microscopy, BioTek, Vermont, USA). Quantitative analysis was performed using flow cytometry.