Immunofluorescence microscopy

Primary hMDMs or THP-1 cells were plated on glass coverslips in a 24-well plate as described above. After 2 h of infection in primary hMDMs or 1 h of infection in THP-1 cells with dsRED-Lp, cells were washed two times with PBS and fixed with 4% paraformaldehyde for 10 min at 37°C. Following fixation, cells were washed and permeabilized with 0.2% Triton X-100 for 10 min. Cells were washed, blocked with 10% BSA for 1 h, and stained with anti-GBP1 or anti-GBP2 primary antibodies for 1 h at RT or overnight at 4°C with anti-galectin-8 primary antibody. Cells were washed with PBS and incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies for 1 h followed by additional washes and mounted on glass slides with DAPI mounting medium (Sigma Fluoroshield).

For anti-GBP1 and anti-GBP2 antibody staining, primary antibodies used were rabbit anti-GBP1 (1:100 dilution; ab131255; Abcam) and mouse anti-GBP2 (1:50 dilution; sc-271568; Santa Cruz). Secondary antibodies were goat anti-rabbit conjugated to Alexa Fluor 488 (4412S; Cell Signaling) and goat anti-mouse conjugated to Alexa Fluor 488 (A11029; Life Technologies) and used at a dilution of 1:4,000. Coverslips were imaged on a Leica SP5 FLIM confocal microscope at 63× magnification, and the percentage of infected cells containing GBP1⁺ or GBP2⁺ intracellular bacteria out of the total number of infected cells was quantified.

For galectin-8 antibody staining experiments, goat anti-galectin-8 (1:200 dilution in primary hMDMs and 1:100 in THP-1 cells; AF1305; R&D Systems) and donkey anti-goat IgG conjugated to Alexa Fluor 488 (1:4,000 dilution; A11055; Invitrogen). Coverslips were imaged on an inverted fluorescence microscope (IX81; Olympus) at a magnification of 60×, and the percentage of infected cells containing galectin-8⁺ LCVs out of the total number of infected cells was quantified.