Characterization of Ferret Immune Cells

Flow cytometry assays were performed as previously described [18 (link)]. Briefly, cells were blocked with Fc blocking antibody (Clone 2.4G2, Cat Nu 553142, BD Biosciences, San Jose, CA) and stained with monoclonal antibodies recognizing ferret CD4 (clone 02 –PE, Sino Biological Inc., Beijing, China), or cross-reacting with ferret CD8 (clone OKT8 –eFluor 450, eBioscience, San Diego, CA), CD11b (clone M1/70 –FITC, eBioscience), anti-MHC class II (clone CAT82A –unconjugated, Kingfisher Biotech, St. Paul, MN), CD3 (clone PC3/188A –FITC and AlexaFluor 647, Santa Cruz Biotechnology, Santa Cruz, CA), and CD79a (clone HM47 –PerCP-Cy5.5, eBioscience). The anti-MHC class II antibody was biotinylated (cat# 130-093-385, Miltenyi Biotec, San Diego, CA) prior to use and detected with streptavidin-eFluor 450 (eBioscience). Unstained cells and isotype controls (eBioscience, San Diego, CA) were included for all antibodies. Data were acquired using a FACSCanto II flow cytometer (BD Bioscience, San Jose, CA), and analyzed using FlowJo software (Tree Star, Ashland, OR).

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Music N., Reber A.J., Kim J.H, & York I.A. (2016). Peripheral Leukocyte Migration in Ferrets in Response to Infection with Seasonal Influenza Virus. PLoS ONE, 11(6), e0157903.

Publication 2016

(Clone 2.4G2, CD11b (clone M1/70 –FITC, streptavidin-eFluor 450 isotype controls FACSCanto II flow cytometer

Alexafluor 647 Anti antibody Antibodies Assays Biological Cd11b Cd79a Cells Clone Cy5 5 Ferret Fitc Flow cytometry Isotype Mhc class ii Monoclonal antibodies Sino Streptavidin Tree

Corresponding Organization : Centers for Disease Control and Prevention

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Variable analysis

independent variables

Fc blocking antibody (Clone 2.4G2, Cat Nu 553142, BD Biosciences, San Jose, CA)
Monoclonal antibodies recognizing ferret CD4 (clone 02 –PE, Sino Biological Inc., Beijing, China)
Monoclonal antibodies cross-reacting with ferret CD8 (clone OKT8 –eFluor 450, eBioscience, San Diego, CA)
Monoclonal antibodies for CD11b (clone M1/70 –FITC, eBioscience)
Monoclonal antibodies for anti-MHC class II (clone CAT82A –unconjugated, Kingfisher Biotech, St. Paul, MN)
Monoclonal antibodies for CD3 (clone PC3/188A –FITC and AlexaFluor 647, Santa Cruz Biotechnology, Santa Cruz, CA)
Monoclonal antibodies for CD79a (clone HM47 –PerCP-Cy5.5, eBioscience)

dependent variables

Percentage or expression levels of cells positive for the measured markers (CD4, CD8, CD11b, MHC class II, CD3, CD79a)

control variables

Unstained cells
Isotype controls (eBioscience, San Diego, CA)

controls

Positive controls: Unstained cells
Negative controls: Isotype controls (eBioscience, San Diego, CA)

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