Flow cytometry analysis
The AQUIOS CL combines robotic automated sample preparation with an
analysis of cells using flow cytometry (27). The AQUIOS CL has one 488nm
diode laser, two light scatter channels (forward scatter and side
scatter), 5 fluorescence channels, and an electronic volume measure.
Absolute leukocyte count was based on an electronic-volume measurement.
A cassette filled with blood tubes was placed in the machine and the
barcodes of the samples were saved. After automatic blood mixing, the
samples were cap-pierced, and 43 μL was pipetted into a 96-deep wells
plate, that is used for antibody staining. The wells plate contained the
bacterial/mitochondrial-derived stimulus
formyl-methionyl-leucyl-phenylalanine (fMLF) (Sigma Aldrich, St. Louis,
MO, USA) with an end concentration of 10-6M. Details
of antibody mixes are provided in the online supplement. Consecutively,
18 μL of a monoclonal antibody mix bound to different fluorescent labels
was added to the 96-wells plate. After 15 min of incubation, the blood
was lysed using 335 μL of lysing reagents A and 100 μL of lysing reagent
B. Lysing reagent A is a cyanide-free lytic reagent that lyses red blood
cells. Lysing reagent B slows the reaction caused by reagent A and
preserved the white blood cells for measurement in the flow cell.
Finally, 100 μL of the prepared sample was aspirated for analysis.
A customized antibody mix was made specifically for this research
purpose. This panel consisted of CD35-FITC (clone J3.D3, Beckman
Coulter, Miami, FL, USA), CD16-PE (clone 3G8, Beckman Coulter),
CD62L-ECD (clone DREG56, Beckman Coulter), CD11b-PC5 (clone Bear1,
Beckman Coulter) and CD10-PC7 (clone ALB1, Beckman Coulter).
The .lmd data files were exported from the AQUIOS CL®and imported into FlowJo® analysis software (Tree Star
Inc., Ashland, OG, USA). The gating strategy is shown in supplemental
figure 1. Granulocytes and monocytes were gated based on forward scatter
and side scatter. Neutrophils and eosinophils in the granulocyte gate
were identified based on positivity or negativity of CD16 expression.
The median fluorescent intensity (MFI) of CD35, CD11b, CD62L and CD10 on
gated neutrophils was used to describe the measure neutrophil and
monocyte responsiveness to fMLF.