Nucleocassette

Peritonitis was induced in mice by ip injection of 0.3 mL of 10% thioglycollate as described (19 (link)). Blood was collected from the tail vein into 3.9% sodium citrate. Peritoneal cells were collected by lavage with 5 mL cold PBS and counted using a hemocytometer. Bone marrow cells were collected by flushing femurs with 5 mL PBS. Blood and bone marrow cells were counted using a NucleoCassette and a NucleoCounter NC-100 (ChemoMetec). For the 11β-HSD1-inhibitor experiments, vehicle (saline with 2% dimethylsulfoxide) or compound UE2316 (10 mg/kg) (21 (link)) was administered by ip injection, the night before and 1 hour prior to thioglycollate injection. Peritoneal cells were collected 4 hours later.

This study collected 50 healthy blood donor buffy coat samples with a negative HIV, HBsAg, and HCV status, which underwent an isolation of pheripheral blood mononuclear cells using Ficoll-Paque™ Plus (GE Healthcare), Dulbecco’s Phosphate Buffered Saline DPBS CTS™ (Gibco life technologies), Fetal Bovine Serum FBS (Sigma) and SepMate™-50 tubes following the manufacturer’s protocol (Stemcell Technologies). The use of anonymized PBMCs from blood donors was conducted in accordance with the rules of the Finnish Supervisory Authority for Welfare and Health (Valvira). Cell count was measured from a mix of 50 µl of cell suspension in DPBS with 2% FBS, 50 µl of Reagent A100 lysis buffer, and 50 µl of Reagent B stabilizing buffer using a NucleoCassette and a NucleoCounter^® NC-100™ (all chemometec). Total RNA was isolated from fresh PBMC samples containing 1–10 × 10⁶ cells using RNeasy Mini kit and Rnase-Free DNAse Set (both Qiagen) within 2h after PBMC isolation. RNA samples were quantified and their purity was assessed with the Qubit™ RNA High Sensitivity Assay Kit in Qubit^® 2.0 fluorometer (ThermoScientific). The RNA quality was checked using an RNA 6000 Pico Kit (Agilent Genomics) in a 2100 Bioanalyzer (Agilent Genomics) to obtain an RNA Integrity Number (RIN) score.