Edta treated vacuum tubes

For white blood cell analysis, mixed blood was collected from heart puncture into EDTA‐treated vacuum tubes (BD biosciences, Franklin Lakes, NJ). Erythrocytes were lysed in hypotonic ammonium‐chloride buffer for 10 min with gentle agitation at 4°C, and then, centrifuged at 250× g for 10 min at 4°C. The supernatant was removed, and pellet was washed twice with cold PBS. The pellet was then resuspended in PBS containing 5 mM of EDTA and 0.5% (w/v) bovine serum albumin (FACS buffer). Purified white blood cells were probed 30 min with the following antibodies: Anti‐CD45‐APC (Cell Signaling Technologies, Danvers, MA), anti‐Ly6G‐FITC (Sigma‐Aldrich, St. Louis, MO), and anti‐CD64‐PE (Thermo‐Fisher). Samples were washed twice with FACS buffer, resuspended in 300 μl of FACS buffer and analyzed immediately.
For platelet analysis, 200 µl of whole blood was collected from the inferior vena cava and added to tubes containing 40 ul of acid‐citrate‐dextrose buffer at 30°C and a portion of blood was aliquoted for counting. Blood was diluted 100x in cold FACS buffer and mixed gently. Diluted whole blood was probed with anti‐CD9‐APC (Thermo‐Fisher) for 30 min. Samples were washed twice with FACS buffer, resuspended in 300 μl of FACS buffer and analyzed immediately.
The Accuri C6 flow cytometer and software (BD Biosciences) was used for all data acquisition and analysis.

Blood samples were drawn into EDTA-treated vacuum tubes (BD, Franklin Lakes, NJ, USA) during treatment procedures. Serum was separated by centrifugation at 2000× g for 10 min. SF was collected in the arthrocentesis procedure from all patients with OA and RA. To remove cells and debris in the SF, the supernatant was collected by centrifugation at 2000× g for 15 min. The viscosity of SF was similar in OA and RA patient groups. Serum and SF were used undiluted for analysis and were stored as 0.5 mL aliquots at −80 °C until analysis.
We obtained 30 serum samples and 30 SF samples (16 paired samples from the same patients) from 45 OA patients, and 29 serum samples and 31 SF samples (13 paired samples from the same patients) from 46 RA patients. SF samples from patients with RA and OA were collected during diagnostic and therapeutic arthrocenteses (for example, intra-articular glucocorticoid or hyaluronic acid injection) of a knee joint. Control serum samples were randomly selected from 30 healthy subjects.