title: Nuocytes
design: Cell Processing.  Previously cryopreserved peripheral blood mononuclear cells (PBMCs) purified from filarial-infected patients seen by the Laboratory of Parasitic Diseases NIAID NIH clinic were compared to PBMCs from normal donors that had also been cryopreserved. PBMCs from both infected individuals and normal donors were thawed, placed over a Ficoll/diatrizoate (LSM, MP Biomedicals, Santa Ana, CA) gradient to remove dead cells, washed twice with serum-free RPMI (Gibco, Grand Island, NY) supplemented with penicillin/streptomycin (Invitrogen, Grand Island, NY), and resuspended in RPMI supplemented with L-glutamine (Invitrogen), penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (Gemini Bioproducts, West Sacramento, CA) for culture.    Cell Sorting. PMBCs isolated from normal blood bank donors, were lineage depleted using the Human Lineage Cell Depletion Kit and the Automacs cell separator (Miltenyi, Auburn, CA).  The negative fraction from the separation was stained with a lineage depletion cocktail in FITC (CD2, CD3, CD14, CD16, CD19, CD56, CD235a) (eBioscience, San Diego, CA) and with anti-CD45-Pacific Blue (eBioscience), anti-cKit- Phyocoerythrin (PE) (eBioscience) and anti-IL-7Rα-Allophycocyanin (APC) (eBioscience).  Sorting for the ILCs was based on being Lineage-/CD45+/cKit+/IL-7Rα+) using a FACS Aria cell sorter (BD Biosciences).  The purity of the sorted cells exceeded 87%.    Cell culture.  Sorted ILC (10,000/well) were put in culture with RPMI 1640 (Gibco) supplemented with penicillin/streptomycin (Invitrogen), L-glutamine (Invitrogen) and 1% human AB serum (Gemini Bio-Products, West Sacramento, CA) with or without a cytokine cocktail containing Il-25 (Peprotech, Rocky Hill, NJ), IL-33 (Peprotech) and IL-7 (Peprotech) (each at 5 mg/ml) and 10U/ml rhuIL-2 (Genentech, San Francisco, CA).  Supernatants were collected every 24 hours and fresh media and cytokines added for up to 5 days.  Cytokine production, IFN-γ, IL-4, IL-5, IL-13, IL-10, and IL-17A, by ILCs was determined by using the Milliplex Max human cytokine panel suspension array technology assays (Luminex™ Millipore, Billerica, MA).  The minimum detectable concentration for the assays varied by cytokine: 0.8 pg/ml for IFN-γ, 1.1 pg/ml for IL-10, 0.7 pg/ml for IL-17, 4.5 pg/ml for IL-4, 0.5 pg/ml for IL-5, and 1.3 pg/ml for IL-13.    Processed PMBCs from filarial-infected patients and normal uninfected controls were cultured at 1 x 106 cells/ml in 24 well plates, with separate wells for ILC quantitation and T-cell quantitation. 10 μl/106 cells of Brefeldin A/Monensin were added at the beginning of the culture.  Cells were incubated for 6 hours at 37oC.  After incubation, cells were harvested using Versine (Invitrogen) treatment as previously described and cells for ILC quantitation resuspended fixed with 4% PFA and in FACS buffer for staining while cells for T cell quantitation were resuspended in FACS buffer without fixation.    RNA Seq. RNA was isolated from sorted ILC (n=6 donors) and lineage depleted PBMCs (control group, n=2 donors) using standard methods as described previously (reference here).  The isolated RNA was cleaned using RNAeasy (Qiagen, Valencia, CA).  The concentration and purity of the RNA was determined by a RNA Pico Chip for the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). Poly-A selected mRNA libraries were constructed from 1 µg total RNA using the Illumina TruSeq RNA Sample Prep V2 Kits (Illumina, San Diego, CA) according to manufacturer’s instructions except where noted.  The cDNAs were fragmented to ~275 bp using a Covaris E210 (Covaris, Woburn, MA). Amplification was performed using 10 cycles to minimize the risk of over-amplification.  Unique barcode adapters were applied to each library.  Equal volumes of individual libraries were pooled and run on a MiSeq (Illumina).  The libraries were then re-pooled based on the MiSeq demultiplexing results.  The pooled libraries were sequenced on two lanes of a HiSeq2000 (Illumina) using version 3 chemistry.  The data were processed using RTA version 1.13.48 and CASAVA 1.8.2. All reads were mapped to available human genome transcripts using in house software developed by J. Ribeiro.  Shortly, the reads were trimmed of low quality ends (cut off = 13) and were blasted against the human transcripts downloaded from the Refseq database of the NCBI (version 12/21/2012) using a word size of 25 and allowing 1 gap extension. Reads were mapped to up to 3 targets if the blast scores were the same.