Apc goat anti mouse igg

Differentiated EBs were dissociated the same way as in the high throughput screening and suspended in 2% FBS/PBS. We analyzed and sorted the CMs using FACSAria Fusion (BD Biosciences), FACSDiva software (BD Biosciences) and FlowJo software. For Ki-67 staining, we used purified mouse anti-Ki-67 Clone B56 (RUO) (BD Biosciences, 556003, 1:200 dilution) and APC goat anti-mouse IgG (minimal x-reactivity) (Biolegend, 405308, 1:500 dilution). To sort the CMs derived from 1390C1 or 409B2, we used PE/Cyanine7 anti-human CD172a/b (SIRPα/β) antibody (BioLegend, 323808, 1:500 dilution), APC mouse anti-human CD90 (BD, 559869, 1:1000 dilution), APC anti-human CD31 antibody (BioLegend, 303116, 1:500 dilution), Alexa Fluor^® 647 anti-human CD49a antibody (BioLegend, 328310, 1:500 dilution) and APC anti-mouse CD140b antibody (BioLegend, 136008, 1:500 dilution).

Nasal epithelial tissues were harvested from pregnant (GD20) and age-matched nonpregnant female rats for flow cytometry experiments. Single cell suspensions were prepared using the following protocol. Tissues were minced and digested with RPMI media supplemented with FCS and Collagenase-IV for 40 min at 37 °C. Cells were filtered through 70-μm filters to remove clumps, washed twice using FACS buffer (2% FCS-PBS) and stained with required antibodies and appropriate controls. Cells were analyzed by flow cytometry using a BD FACS Canto II instrument (BD Biosciences, San Jose, CA) and data were analyzed using FlowJo (BD Biosciences Inc.). The following antibodies were used: FITC∗SNA-I (Sambucus nigra agglutinin-I; #F-6802-1, EY Laboratories Inc., San Mateo, CA), Biotin∗MAA (Maackia amurensis agglutinin; #BA-7801-2, EY Laboratories Inc., San Mateo, CA), Brilliant Violet 421∗Streptavidin (#405226, BioLegend, Inc.), mAb-EpCAM (GZ-1) (#ab187276, Abcam plc., Cambridge, MA) and APC Goat anti-mouse IgG (#405308, BioLegend, Inc.). Dead cells were excluded from analysis by staining with LIVE/DEAD Fixable Blue Dead Cell stain kit (#L23105, ThermoFisher Scientific Inc.). Live, singlet, EpCAM⁺ and CD45^- cells were considered as epithelial cells, upon which the expression of SNA-I and MAA were scored.