ASC protein aggregates (specks)

Extracellular ASC Speck detection by flow cytometry

Assay Protocol

Materials                 

Phycoerythrin conjugated anti-ASC (TMS-1) Antibody (clone: HASC-71, stored at 4°C) 

Non-fluorescent 1-µm microspheres (F13838, Thermo Fisher Scientific)

Serum/supernatant (stored at -80°C) 

Method

Sample preparation

1. Samples from patients and healthy controls were collected in VACUETTE® tubes (Greiner Bio-One), coated with EDTA anticoagulant or serum clot activator gel for whole blood or serum, respectively. Serum samples were collected by allowing the sample to clot for 1 hr, followed by centrifugation at 1100 x g for 10 min and subsequent storage at -80 ⁰C. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Lymphoprep (Axis-Shield, Dundee, UK). Monocytes were isolated by negative selection from PBMCs using the monocyte isolation kit II (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
2. Monocytes (1 x 10⁶ / mL) were stimulated with LPS (10ng/ mL Ultrapure EK, Invivogen) for 4 hr with the addition of ATP (5 mM, Invivogen, San Diego, California) for the final 30 min of stimulation to prime/ activate the NLRP3 inflammasome. 
3. For the assay, 200 µL serum/ media was transferred into FACS collection tubes, and incubated with 5 µL of phycoerythrin anti-ASC (TMS-1) antibody (HASC-71 clone, Biolegend, 653904) for 1 hour at 4^oC. Ensure the serum and antibody are at room temperature prior to use. Aliquot an additional 200ul of each serum or media into fresh tubes which will be negative control (no antibody).

Flow cytometry

1. Using a LSRII flow cytometry instrument (BD Biosciences), a template was set up to measure extracellular ASC specks. Size gating was carried out using Non-fluorescent 1-µm microspheres (F13838, Thermo Fisher Scientific), according to manufacturers’ specifications to generate a size guide for ASC specks using FSC vs SSC density plot. A gate was drawn around the non-fluorescent 1-µm microspheres to measure the ASC specks.  A total of 200ul of negative control was run within the 1um gate. 
2. A single parameter histogram plot with phycoerythrin filter vs count was generated to specifically identify ASC positive particles. ASC specks were reported as total events in the set gate. The negative control (no antibody) were used to set the threshold of negatively stained particles, and positively stained ASC specks recoreded above the threshold. The total positive events in the set gate was divided by 200 to give ASC speck per microliter.
3. The unstimulated samples were used as controls for the experiment and percentage change recorded for the LPS/ATP treated samples.