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.