Flow Cytometry analysis
Freshly processed nasal lavage cells were divided into 0.2 to 1 x
106 cells per tube, resuspended in 50 µl of
phosphate-buffered saline (PBS) (Lonza) and incubated in the presence of
antibodies for 20 minutes on ice in the dark. Cells were then washed
with PBS and resuspended for analysis. The following antibodies to human
cell membrane proteins were used: CD45-PE Cy7, CD14-APC eFluor,
CD16-FITC, CD56-APC, CD3-PerCPCy5.5 (all from eBioscience, San Diego,
CA) and NKp46-PE (9E2) (BioLegend). Data was acquired on an ARIA flow
cytometer (Becton Dickinson) with FACSDiva acquisition software (Becton
Dickinson BioSciences, Mississauga, Ontario, Canada) and analyzed using
FlowJo flow cytometric analysis software version 10.1 (Tree Star,
Ashland, OR). Leukocyte populations were identified in nasal lavage
samples as described previously for induced sputums (17). Briefly,
doublets were excluded by gating on forward scatter (FSC)-H and FSC-A,
followed by gating on CD45 positive cells to discriminate leukocytes
from contaminating squamous-epithelial cells. Cellular debris were then
gated out on the basis of FSC and side scatter (SSC). Specific
populations were identified on the basis of marker expression as well as
size and granularity. Respective gating strategy is outlined in Fig 2A.
For nasal lavage samples, as many events as possible were collected. For
compensation control, BD CompBeads (BD PharMingen) were used.