Flica 660 caspase 1 assay kit

A pool of 3 dpf Tg(mpeg1::Gal4-FF)^gl25/Tg(UAS::LIFEACT-GFP)^mu271 larvae were infected systemically with 10000 CFUs of mCherry–S. sonnei 53G, mCherry–S. flexneri M90T, or PBS containing mock solution. At 4 hpi, larvae were dissociated by treatment with 1 mL of 4% trypsin for 15 minutes at 28 °C. Single-cell dissociation was facilitated by mechanical disruption with a P1000 pipette after trypsin treatment. Dissociated cells were harvested by centrifugation (5 minutes, 800g, room temperature), washed in calcium-free PBS, separated by passage on a 4 μm cell strainer, and suspended in 500 μL of staining solution for active caspase 1 (FLICA 660 Caspase-1 Assay Kit, probe 660-YVAD-FMK, No. 9122; ImmunoChemistry Technologies) prepared per the manufacturer's guidelines. Upon staining, cells were pelleted, washed in PBS, and fixed in 4% paraformaldehyde overnight. For flow cytometry, cells were washed with 2 mL of PBS twice and resuspended in 300 μL of PBS. Active caspase 1 staining of single macrophages was measured on a LSRII (BD Biosciences) and data were analyzed with FlowJo version 10.7.1.

Protein A-Sepharose and Protein G-Sepharose CL-4B Fast Flow beads were from GE Healthcare Bio-Sciences Corp., Piscataway, NJ. Cyto Nuclear extract kit was from Active Motif, Carlsbad, CA. Trichostatin A (TSA), Leptomycin B (LPT), and nicotinamide were from Sigma-Aldrich. The CytoTox 96 non-radioactive cytotoxicity kit was from Promega, Madison, WI. SlowFade Gold Antifade reagent with DAPI was from Life Technologies. Verikine human IFN-β ELISA kit was from PBL Assay Science, Piscataway Township, NJ. IL-1β ELISA kit was from RayBiotech, Inc. Norcross, GA. The FLICA 660 Caspase-1 Assay kit was from Immunochemistry Technologies, Bloomington, MN. P300 and HDAC activity assay kits were from BioVision Inc., Milpitas, CA.