Fam flica reagent

THP-1 cells (1 × 10⁶ cells per ml) were pre-treated with BC-1471 (10 μg ml⁻¹) for 16 h prior to addition of LPS (500 ng ml⁻¹), Pam3CSK4 (100 ng ml⁻¹) or control for 6 h. FAM-FLICA reagent (Immunochemistry Technologies, 98) was added 4 h before the end of the assay, prepared according to the manufacturer's protocol. Cells were washed and resuspended in wash buffer, followed by flow cytometry analysis using the 488 nm excitation channel.

Mice were inoculated with 3 x 10⁴ PFU HSV-1 KOS or mock-infected and sacrificed by transcardial perfusion with PBS at day 3 post-infection. Brains were dissociated using the Neural Tissue Dissocation Kit (P) (Miltenyi Biotec). For enumeration of infiltrating leukocytes, cells were first treated with TruStain FcX anti-CD16/32 antibody (Fc block-BioLegend; 93) before staining with the following antibodies: Ly6G APC-Cy7 (BioLegend; RB6-8C5), CD11c APC (BioLegend; N418), and CD11b BV421 (BD; M1/70). Flow cytometry was performed on a FACS CantoII (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo 10.1 software (Ashland, OR, USA).
For fluorescently-labeled inhibitor of caspase activity (FLICA) assays, cell suspensions were first incubated with a 1:1000 dilution of Live/Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) before incubation with the FAM-FLICA reagent according to the manufacturer’s protocol (ImmunoChemistry Technologies). Cells were treated with an Fc-blocking reagent (Fc block-BioLegend; 93) and stained with the following antibodies before being analyzed: ACSA-2 PE (Miltenyi Biotec; REA969), CD45 PerCP-Cy 5.5 (BD Biosciences; 30-F11), and CD11b PE-Cy7 (eBioscience; M1/70). Gating of positive populations was based on Fluorescence minus one control (FMO) samples.