Fam flica caspase 1 assay kit

To assess pyroptosis in HSCs, caspase-1 activation was quantified with a FAM-FLICA Caspase-1 Assay Kit (ImmunoChemistry Technologies, Bloomington, USA) following the manufacturer’s instructions. Briefly, after stimulation with SEA for 24 h, the cells were harvested and incubated with caspase-1 detection probe for 1 h in the dark. After the unbound FLICA reagent was removed with washing buffer, the cells were stained with propidium iodide (PI) for 20 min and then analysed with flow cytometry (BD FACSCalibur, Franklin Lakes, USA). Pyroptotic cells were defined as cells that were double positive for activated caspase-1 and PI. For ROS detection, HSCs were cultured on well plates, loaded with DCFH-DA (20 μM) in serum-free medium in the dark for 30 min, and washed three times with wash buffer. The mean fluorescence intensity of intracellular ROS was examined with a FACSCalibur flow cytometer, and the results were analysed with FlowJo software (TreeStar, Ashland, USA)

Active caspase-1 was detected using a fluorescent inhibitor of caspases (FAM-FLICA Caspase 1 Assay Kit, Immunochemistry Technologies, Bloomington, Minnesota) according to the manufacturer’s instructions. First-passage human goblet cells were grown in 96-well plates. Before use media was changed to antibiotic-free RPMI media containing 1% FBS. The cells were then were stimulated with bacteria for 4 hours and 10 µL of 30X FLICA solution added. Following incubation, cells were stained with Hoechst 33 342 stain (0.5% w/v) and viewed on an inverted phase-contrast microscope equipped for epifluorescence (Eclipse TE300, Nikon, Tokyo, Japan) with a UV filter with excitation 490 nm, emission >520 nm for green fluorescence for caspase-1 positive cells and excitation 365 nm, emission 480 nm for visualisation of nuclei stained with the Hoechst dye. The total number of nuclei in four ×400 fields of view was counted, and the number of cells staining green (indicated active caspase-1) was expressed as a percentage of the total number of cells. For each experiment, an exposure time was set for the basal condition and held constant for the conditions in which bacteria were present.

Presence of activated caspase-1 was quantified by flow cytometry (MACSQuant Analyzer 8, Miltenyi Biotec, Bergisch Gladbach, Germany) using the FAM-FLICA® Caspase-1 Assay Kit (ImmunoChemistry Technologies, LLC, Bloomington, MN, USA) as previously described [14 (link)]. Briefly, whole blood was aliquoted and stimulated with either LPS (10 ng/mL final concentration in culture) or left unstimulated (control) for 60 min at 37 °C and 5% CO₂ in the dark. The fluorescent inhibitor probe FAM-FLICA® (consisting of FAM-YVAD-FMK) was added to the culture to stain active caspase-1, and cultures were incubated for a further 50 min. To identify monocytes, cluster of differentiation (CD)14 VioBlue (Miltenyi Biotech) was then added during the final 10 min of incubation. After a total of 120 min of incubation, red blood cells were lysed, and two washes were conducted to remove red blood cell debris. Propidium iodide was added to stain dead cells, and samples were immediately analyzed by flow cytometry. A hierarchical gating strategy was used to acquire data. Flow cytometry data were analyzed using MACSQuantify^TM (Version 2.6, Miltenyi Biotec, Bergisch Gladbach, Germany).

Alexa Fluor™ 488 Protein Labelling Kit, E. coli BL21 Star™ (DE3) strain, isolectin GS-IB₄ from Griffonia simplicifolia conjugated with Alexa Fluor488 or Alexa Fluor568, IL-18 (Rat) ELISA kit, pHrodo™ red E.coli BioParticles conjugate, were purchased from Invitrogen, ThermoFisher Scientific (USA). Cell culture reagents DMEM Glutamax, fetal bovine serum, horse serum, penicillin–streptomycin, Versene (1:5000) solution were from Gibco, ThermoFisher Scientific (USA). Poly-(l)-lysine was from R&D systems (USA), IL-1β (Rat) ELISA kit from Abbexa (United Kingdom), FamFlica Caspase-1 Assay Kit from ImmunoChemistry Technologies (USA), anti-TLR4 antibody from Santa Cruz Biotechnology (USA). Cell permeable Ac-YVAD-CHO, VX-765, GSK2795039, MCC950, and IgG were obtained from Merck (Germany). All other materials were purchased from Sigma-Aldrich (USA).

SH-SY5Y cells were seeded in 6-well plates at 10⁶ cells/well for 24 h and then treated with increasing MPP⁺ concentrations for 24 h. Cells were gently washed with PBS and chemically detached with 0.05% trypsin/EDTA. After detachment, trypsin was quenched with complete media, cells were pelleted by centrifugation, and the caspase activity was detected with the FAM-FLICA caspase-1 assay kit (ImmunoChemistry Technologies, LLC, Bloomington, MN, USA) according to the manufacturer’s recommendations. Briefly, cells were stained and incubated with FLICA (1:30) for 1 h at 37 °C protected from light. Cells were then by washed with 5 volumes of 1× wash buffer, pelleted by centrifugation, resuspended in 1× apoptosis wash buffer with 5 µL of propidium iodide (PI), and incubated for 10 min at 37 °C protected from light. Data were acquired with a Beckman DxFlex flow cytometer (Beckman, Brea, CA, USA) and analyzed with CytExpert (Beckman Coulter Inc, CA, USA). Single stain controls were used to calculate the “compensation,” which refers to the process of correcting fluorescence spillover, that is, removing the signal of any given fluorochrome from all detectors except the one devoted to measuring that dye. PI-positive cells were detected according to the operating procedure of PI-staining kit (KeyGEN Biotech, Nanjing, China)