Table 2.

Primary antibodies and secondary reagents used for FCM analyses.

Antigen	Clone	Isotype	Fluorochrome	Labeling strategy	Source of primary Ab
Ex vivo phenotyping
NK cells
CD3	BB23-8E6	IgG2b	Alexa647	Secondary antibodya)	Southern Biotech
CD8α	11/295/33	IgG2a	BV421	Biotinb)-streptavidinc)	In-house
CD16*	G7	IgG1	PE	Secondary antibodyd)	Bio-Rade)
NKp46*	VIV-KM1	IgG1	PE	Secondary antibodyd)	In-house
NKp44*	54-1	IgG1	PE	Secondary antibodyd)	In-housef)
perforin*	Pf344	IgG1	PE	Secondary antibodyd)	Mabtechg)
pDC panel I
CD4	74-12-4	IgG2b	FITC	Directly conjugated	BD Biosciences
CD172a	74-22-15A	IgG2b	BV421	Biotinb)-Streptavidinc)	In-house
CD16*	G7	IgG1	PE	Secondary antibodyd)	Bio-Rad
NKp46*	VIV-KM1	IgG1	PE	Secondary antibodyd)	In-house
NKp44*	54-1	IgG1	PE	Secondary antibodyd)	In-housef)
perforin*	Pf344	IgG1b	PE	Secondary antibodyd)	Mabtech
pDC panel II
CD4	74-12-4	IgG2b	FITC	Directly conjugated	BD Biosciences
NKp46	VIV-KM1	IgG1	Alexa647	Directly conjugatedh)	In-house
CD14*	CAM36A	IgG1	PE	Secondary antibodyd)	Kingfisher Biotech
CD16*	G7	IgG1	PE	Secondary antibodyd)	Bio-Rad
CD163*	2A10/11	IgG1	PE	Directly conjugated	Bio-Rad
CD172a*	74-22-15	IgG1	PE	Secondary antibodyd)	In-house
Intracellular cytokine staining
pDCs
CD4	74-12-4	IgG2b	Alexa488	Secondary antibodyi)	In-house
CD172a	74-22-15	IgG1	eFluor450	Biotinb)-streptavidinj)	In-house
NKp46	VIV-KM1	IgG1	PE	Secondary antibodyd)	In-house
IFN-α*	F17	IgG1	Alexa647	Directly conjugatedh)	Thermo Fisher Scientific
IFN-γ*	CC302	IgG1	Alexa647	Directly conjugated	Bio-Rad
TNF-α	MAb11	IgG1	BV605	Directly conjugated	BioLegend
Phospho-specific staining unsorted cells
NK cells
CD3	BB23-8E6	IgG2b	Alexa647	Secondary antibodya)	Southern Biotech
CD8α	11/295/33	IgG2a	eFluor450	Biotinb)-Streptavidink)	In-house
pS6(S235/S236)	cupk43k	IgG1	PE	Directly conjugated	Thermo Fisher Scientific
pDCs
CD4	74-12-4	IgG2b	Alexa488	Secondary antibodyk)	In-house
CD172a	74-22-15	IgG1	Alexa647	Directly conjugatedh)	In-house
pS6(S235/S236)	cupk43k	IgG1	PE	Directly conjugated	Thermo Fisher Scientific
FACS Sort
CD4	74-12-4	IgG2b	PerCP-Cy5.5	Directly conjugated	BD Biosciences
CD8α	11/295/33	IgG2a	BV421	Secondary antibodyk)	In-house
CD172a	74-22-15A	IgG2b	Alexa647	Biotinb)-Streptavidinl)	In-house

^aGoat anti-mouse anti-IgG2b-Alexa647, Thermo Fisher Scientific.

^bEZ-Link™ Sulfo-NHS-LC-Biotin, Thermo Fisher Scientific.

^cStreptavidin-Brilliant Violet 421, BioLegend, San Jose, CA, USA.

^dGoat anti-mouse anti-IgG1-PE, Southern Biotech, Birmingham, AL, USA.

^eBio-Rad, Hercules, CA, USA.

^fKindly provided by Joan K. Lunney, Animal Parasitic Disease Laboratory, BARC, ARS, USDA, Beltsville, MD, USA (28).

^gMabtech, Nacka Strand, Sweden.

^hAlexa Fluor™ 647 Antibody Labeling Kit, Thermo Fisher Scientific.

ⁱGoat anti-mouse anti-IgG2b-Alexa488, Thermo Fisher Scientific.

^jStreptavidin-eFluor450, Thermo Fisher Scientific.

^kGoat anti-mouse anti-IgG2a-Brilliant Violet 421, Jackson ImmunoResearch, Suffolk, UK.

^lStreptavidin-Alexa647, Thermo Fisher Scientific.

*Used in different samples.