8 protocols
ID_REF = VALUE = normalized
Phycoerythrin fluorescence was quantified with an Affymetrix scanner, and Affymetrix Command Console software was used to generate cel files from the raw scan data.
Cel files were quantile-normalized with the GC-RMA method using the open-source gcrma Bioconductor package (R programming language).
Biotinylated cDNA was hybridized overnight with Affymetrix U133_Plus_2.0 arrays using conditions recommended by NuGEN. After washing, arrays were stained with SAPE followed by signal enhancement with anti-SAPE antibody as recommended by Affymetrix.
Total RNA was used to prepare biotinylated cDNA with the WT-Ovation Pico kit as recommended by the kit manufacturer (NuGEN).
CFSE-stained cells were harvested, washed, and counted. The cells were stained with Live/Dead Violet Invitrogen, Carlsbad, CA), washed and stained with anti-CD27-APC-H7 antibody (BD Bioscience, San Diego, CA) on ice. FACS cell sorts were performed on live cells.
Freshly isolated CD27+ IgG enriched human peripheral blood Bc were cultured in the presence of CpG 2006 (10 ng/ml, Oligos, etc., Wilsonville, OR), plus recombinant human cytokines IL-2 (20 IU/ml), IL-10 (50 ng/ml), IL-15 (10 ng/ml) (all from BD Biosciences, San Diego, CA), and recombinant human BAFF (75 ng/ml, Chemicon, Temecula, CA) PC-L medium (IMDM medium, lacromin (50mg/ml, Seracare, Milford, MA), insulin (5mg/ml, Sigma-Aldrich, St. Louis, MO), penicillin/streptomycin (1x, Invitrogen, Carlsbad, CA), gentamicin (15mg/ml, Invitrogen), heat inactivated fetal bovine serum (10% v/v, Invitrogen), normocin (0.1% v/v, Invivogen, San Diego, CA) in round-bottomed 96-well plates (BD Biosciences, San Diego, CA). All cells were cultured at 37 C, 5% CO2.
Immediately after collection, sorted cells were lysed in RLT buffer and passed through a Qiashredder column (both from Qiagen, Germantown, MD) and snap frozen in liquid nitrogen. Samples were stored at -70C until RNA extraction. RNA was extracted using the RNeasy Micro Kit with on-column DNAse as recommended by the manufacturer (Qiagen).