Vacutainer cpt mononuclear cell preparation tube sodium citrate

Blood was collected in 2.5 ml PAXgene™ Blood RNA Tubes (PreAnalytiX, Qiagen, Valencia, CA, USA), in heparin or sodium citrate anticoagulant coated tubes (BD Vacutainer^® CPT™ Mononuclear Cell Preparation Tube—Sodium Citrate, BD Biosciences, San Jose, CA, USA) for peripheral blood mononuclear cell (PBMC) isolation. Total RNA was extracted using the column based method kits of PreAnalytix (Qiagen) for PAXgeneTM tubes and RNeasy (Qiagen) for PBMC isolation from Heparin coated tubes as per manufactures protocol. On-column DNAse treatment was performed to eliminate genomic DNA contamination in all samples. PBMC from sodium citrate coated CPT tubes were separated as described in the product insert for BD Vacutainer CPT, washed, and counted. PBMCs were subsequently resuspended in 90% fetal bovine serum (FBS; Gemini, Sacramento, CA, USA) and 10% dimethylsulfoxide (DMSO; Sigma, St. Louis, MO, USA) solution, and progressively cooled overnight to −80°C and subsequently transferred to liquid nitrogen or −150°C for storage. Quantity of total RNA was measured by NanoDrop spectrophotometer (NanoDrop).

Serum samples and PBMCs were collected between month 1 (June 2014) and month 11 (April 2015). For serum collection, whole blood was collected in serum separator tubes (SST). Tubes were spun at 3000 rpm for 10 min at room temperature. Serum was collected and placed at -80°C for future use.
For PBMC isolation, blood samples were collected in BD Vacutainer CPT Mononuclear Cell Preparation Tube-Sodium Citrate (BD Biosciences, 362761). Tubes were spun at 1500 x g at room temperature for 30 min. Samples were then shipped at 4°C overnight. Upon arrival, the plasma layer was removed from the top of the gel plug and diluted with an equal volume of DPBS. The sample was spun at 1200 rpm for 10 min. The supernatant was removed and RBCs were lysed. After RBC lysis and a wash with PBS, the final cell pellet was resuspended in cDMEM (Gibco, 11995–065) with 10% FBS, L-glutamine (Gibco, 25030–081), Antibiotic-Antimycotic Solution (Sigma, A5955), and MEM NEAA (Gibco, 11140–050).