Magnetic nanoparticles

CD4 and CD8 T cells were depleted from PBMC by positive selection using EasySep positive selection cocktail containing monoclonal antibodies coupled to Magnetic Nanoparticles (Stemcell Technologies, Vancouver, BC, Canada). After the cell separation total CD4 and CD8 T cell subsets were assessed by flow cytometry. Briefly, 1,000,000 cells were stained in 5 mL Falcon round bottom tubes with human anti-CD4 FITC and CD8 PE (all BD Biosciences, San Jose, CA, USA) and fixed in 2% formaldehyde. A minimum of 200,000 events were acquired using BD FACSCanto III (BD) and analyzed with FACSDivaTM, version 4 software.

Twenty five μL of anti-CD45 antibody tetramer (MEM-28 Clone, CD45 Depletion kit, StemCell Technologies, Inc.) was added to the cells suspended in 1 mL of Separation Buffer and incubated for 30 minutes, at RT in a rotator. Subsequently, 50 μL of magnetic nanoparticles (Stem Cell Technologies, Inc.) was added, and the incubation continued for another 15 minutes. The volume was made up to 5.5 mL with Separation Buffer. This cell sample was referred to as “Feed”.

DNA was isolated from whole blood in all patients. In six, we were able to obtain additional samples, including saliva, buccal epithelial cells, urine, fresh blood for isolation of circulating lymphocytes, and myeloid cells. Isolation of neutrophils, monocytes, and T and B lymphocytes was performed with magnetic nanoparticles (Stemcell Technologies, Inc., Manchester, UK) following the manufacturer’s protocol. DNA was extracted from isolated cells using Qiagen Investigator DNA extraction Kit (Qiagen Ltd., Manchester, UK).
NLRP3/CIAS1 (exons 3, 4, and 6) [NCBI RefSeqGene NC_000001.10 (LRG_197)] and NLRP12 (exon 3) [NCBI RefSeqGene NC_000019.9] genes were amplified by PCR and analyzed by Sanger sequencing using the protocol described previously (16 (link)). The chromatograms were analyzed on the ABI 3130xl Genetic Analyser using Sequencing Analysis Software version 5.4.
Amplicon-based deep sequencing was performed to amplify all exons of the NLRP3 gene using specific pair primers as previously described (17 (link)). All PCR amplicons were deep sequenced (mean coverage 3,500×) on different platforms (GS Junior 454, PGM IonTorrent and Illumina MiSeq). The sequences were analyzed using the Amplicon Variant Analyzer software (Roche, Switzerland).