FC analysis
Cells were resuspended in a concentration of 3X106cells/ml and 3X105 cells/100µl were seeded per well of
a 96 U-bottom plate. Cells were centrifuged (150g 5 min, 4°C),
resuspended and incubated in 100µl blocking buffer (FC buffer+5% goat
serum) (15 min on ice). Thereafter, cells were centrifuged, resuspended
and incubated with the following antibodies for 40 min: CD117 (c-Kit)
monoclonal antibody (2B8), APC (# 17-1171-82, ThermoFisher Scientific,
Waltham, MA, USA), PE anti-mouse FcεRIα antibody (#134308, BioLegend,
Inc., San Diego, CA, USA), PE Rat Anti-Mouse CD170 (Siglec-F)
(#552126,BD), APC anti-mouse CD193 (CCR3) antibody (144512, BioLegend,
Inc.), PE anti-mouse F4/80 antibody (#123109, BioLegend, Inc.), CD11b
monoclonal antibody (M1/70), APC (# 17-0112-82, ThermoFisher
Scientific). All antibodies were matched with their corresponding
isotype control: APC Rat IgG2b, κ Isotype Ctrl Antibody (#400612,
BioLegend, Inc.), Armenian Hamster IgG Isotype Control (eBio299Arm), PE
(# 12-4888-83, ThermoFisher Scientific.), PE Rat IgG2a, κ Isotype Ctrl
Antibody (400508, BioLegend, Inc.), APC Rat IgG2a, κ Isotype Ctrl
Antibody (400512, BioLegend, Inc.). After incubation, cells were washed
twice with FC buffer, centrifuged and resuspended in 200µl FC buffer and
acquired in a BD™ LSR II flow cytometer.