Calibur system

Flow cytometry analysis was performed three days
after transfection. The cells were washed twice with
KO-DMEM, dissociated with trypsin, then centrifuged
and resuspended at 1×10⁶ cells/ml in PBS-. The
cells were stored at 4˚C for a maximum of 1 hour before
analysis. Acquisition was conducted on a fluorescence-
activated cell sorting (FACS) Calibur system
(BD Biosciences, Heidelberg, Germany) and sample
analyses were carried out by CellQuest software (BD
Biosciences, Heidelberg, Germany). The gating criteria
for analysis of the EGFP expressing cells were
set according to the level of auto-fluorescence of a
non-transfected control.

The procedure for staining the cell surface proteins was previously described [26 (link)]. Monocytes were first gated according to their FSC and SSC dot plots, and then three-color fluorescence was measured within the monocyte gate. Monocytes were subclassified according to CD14 and CD16 expression. The CD14⁺CD16⁻ (Mon1) cells were defined as monocytes expressing CD14 but not CD16. CD14⁺CD16⁺ monocytes were then further subclassified regarding CCR2 expression into CD14⁺⁺CD16⁺CCR2⁺ (Mon2) and CD14⁺CD16⁺⁺CCR2⁻ (Mon3). At least 20,000 events in the monocyte gate were measured per experiment. The results are presented as the percentages of positive cells. Circulating MPA was defined by double positivity for CD14 and CD42a. Intracellular staining of cytokines was performed using a BD Cytofix/Cytoperm Fixation and Permeabilization kit (BD Biosciences). Appropriately conjugated IgG antibodies were used as isotype controls. After washing with phosphate-buffered saline (PBS), the stained cells were subjected to flow cytometry analysis using a fluorescence-activated cell sorter (FACS) Calibur system and Cell Quest software (BD Biosciences).