Monocyte profiling
CD14/CD16 monocyte profiling was carried out by flow cytometry. Briefly, cryopreserved mononuclear cells were rapidly thawed under warm water, washed with complete medium and resuspended in staining buffer. For fresh samples, mononuclear cells from heparinized peripheral blood or bone marrow samples were obtained by density gradient separation using Fico/Lite-LymphoH (Atlanta Biologicals; Flowery Branch, GA), washed in complete medium (RPMI1640 + 10% fetal bovine serum + gentamycin) and re-suspended in phosphate-buffered saline (PBS) + 30% adult bovine serum as a blocking agent for non-specific staining of immunoglobulins. Cell viability was assessed by trypan blue staining. Surface markers were assessed for 3-color immunophenotyping by incubating mononuclear cells for 20 minutes in the dark with fluorescence-conjugated monoclonal antibodies against CD45 (PC5), CD16 (FITC) and CD14 (PE) (Beckman Coulter; Brea, CA), followed by washing in PBS and re-suspension in PBS + 0.4% formaldehyde as a fixative prior to acquisition using a Coulter XL-MCL flow cytometer (Beckman Coulter; Brea, CA) equipped with an Argon laser. Monocyte populations were gated based on CD45/SS and CD14/SS analysis. Thereafter, CD14/CD16 double parameter plots were used to further define the three subsets of mature monocytes, CD14++/CD16- classical (MO1), CD14+/CD16++ intermediate (MO2), and CD14-/CD16++ non-classical (MO3) monocytes.6-9 The distribution of monocytes was reported as percentages within the total monocyte gate.