Computational protocol: Genetic Diversity in the Collaborative Cross Model Recapitulates Human West Nile Virus Disease Outcomes

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Protocol publication

[…] Following preparation of single-cell suspensions, cells were plated at 1 × 106 cells/well and stained for surface markers for 15 min on ice. For tetramer staining, cells were stained with the WNV NS4b-H2Db tetramer (generated by the Immune Monitoring Lab, Fred Hutchinson Cancer Research Center). Cells were subsequently fixed, permeabilized (Foxp3 fixation/permeabilization concentrate and diluent; eBioscience), and stained intracellularly with antibodies for 30 min on ice. Flow cytometry was performed on a BD LSRII machine and with BD FACSDiva software. Analysis was performed using FlowJo software.The following directly conjugated antibodies were used: CD3-ECD (143-2C11), CD4-BV605 (RM4-5), CD8-BV650 (53-6.7), Foxp3-Alexa700 (FJK-16S), and NS4b class I tetramer-allophycocyanin. The Foxp3 intracellular staining kit (eBioscience) was used for fixation/permeabilization and all intracellular staining. AmCyan Live/Dead stain (Invitrogen) was used in all panels for identification of live cells. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Databases H2DB
Application Flow cytometry
Organisms Mus musculus, West Nile virus, Homo sapiens
Diseases Infection, Virus Diseases, HIV Infections