Operetta high throughput screening

The knockout experiment was carried out in M1 macrophages from RAW264.7 cell. TrueCut™ Cas9 Protein v2 (Invitrogen™, A36498) and Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (Invitrogen™, CMAX00008) were utilized according to the manufacture’s guideline. The sgRNA in this experiment was purchased from Horizon (Target ID, SG-044254-01-0005; Target sequence, 5′-GGATGGGCTCTCCGTAGCGG-3′). The images were taken by Operetta High Throughput Screening (Perkin Elmer, Walthan, MA, USA), and the data were collected from Harmony 4.8 (Perkin Elmer). The fluorescent intensity was measured by the ImageJ (1.53) software, placing 10 regions of interest in randomly selected areas. The separate three experiments were proceeded with similar results. No data were excluded from the analyses.

For the cell screening, M0, M1, and M2 from RAW264.7 cells were seeded into 96 well plates, and incubated with 1 μM of LC library in duplicate at 37 °C. After 1 h, cells were washed with PBS one time, and Operetta High Throughput Screening (Perkin Elmer, Walthan, MA, USA) was used to acquire the images from the screening. The data were collected from Harmony 4.8 (Perkin Elmer) and the fluorescent intensity was measured by the ImageJ (1.53) software, placing 10 regions of interest in randomly selected areas.

For the characterization of differentiated macrophages, APC anti-mouse CD86 Antibody (dilution 1:100, BioLegend, 105012), anti-mouse CD206 antibody (1:100, BIO-RAD, MCA2235) conjugated with Goat anti-Rat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:500, Invitrogen, A-11006), Human CD38 Alexa Fluor® 488-conjugated Antibody (1:100, R&D Systems, FAB2404G), and CD36 Monoclonal Antibody (1:100, Invitrogen, MA5-14112) conjugated with Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (1:500, Invitrogen, A32723) were incubated with the cells for 30 min, and Hoechst33342 (1 μg/mL) was added for 5 min prior to the experiment. The images were taken by Operetta High Throughput Screening (Perkin Elmer, Walthan, MA, USA), and the data were collected from Harmony 4.8 (Perkin Elmer).