Three and sometimes four blood pressure determinations were collected by a physician using a mercury sphygmomanometer.
Blood samples for serum EPO concentration determinations were collected by venipuncture, before and 2, 6, 10, 14, 17, 20, 24, 48, 72, 96, and 120 hours after injection.
Blood for plasma determinations was collected by cardiac puncture and anticoagulated in the presence of trisodium citrate (3.15% w:v, 1 voL citrate per 9 vol blood).
The animals received daily injections of 17 ß-estradiol (E2) (1 μg/kg, group E, n = 4), progesterone (P) (0.3 mg/kg, group P, n = 4) or corn oil vehicle (0.1 ml/kg group C, n = 3) i.m. on Days 0, 1 and 2. Blood samples for P4 and E2 determinations were collected daily by jugular venipuncture before, during and after the treatment, and on Day 3 all lambs were slaughtered.
For apoptosis determination cells were collected by trypsinization into their culture media, washed with PBS and stained with Annexin V- Alexa Fluor 488 (InVitrogen) and propidium iodide (PI), and analyzed immediately after 15 minute incubation using the CellQuest software on the FACScan (Becton Dickinson).
For TNF-α determination, the cell-free supernatants were collected by centrifugation and frozen at −80°C until determination of mediator content by the use of a specific ELISA kit according to manufacturer's instructions (Coulter, France).
For determination of protein content, cells were collected by trypsinization and lysed with mammalian protein extraction reagent (Thermo Scientific, Rockford, IL, USA) and complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA) at 4°C for 30 minutes.
For CDM determination, more than 9 × 10 cells were collected by centrifugation, washed with Dulbecco's PBS and dried as described above.
Blood samples were collected by venicpuncture for determination of serum bone specific alkaline phosphatase (BSAP) activity (ALKPHASE-B, Meta-Biosystems Inc, Mountain View, CA) with sensitivity of 0.7 U/L, intra- and inter assay CV of 5.2 and 5.8%, respectively.
For biomass determination, 5 to 10 mL of culture samples were collected by centrifugation, washed twice with RO-H2O, and dried to constant weight at 80 °C.
For determination of cell dry weight, different volumes (0.5 ml - 1.5 ml) of cell cultures were collected by centrifugation.
