infected with: none (mock control) tissue: spleen days post infection (dpi): 0
Growth protocol
Twenty juvenile pigtailed macaques (Macaca nemestrina) were dual-inoculated with the molecular clone SIV/17E-Fr and the immunosuppressive swarm SIV/DeltaB670. Groups of six infected macaque were euthanized at 4, 7, and 14 days post infection (p.i.), and two animals were euthanized at 21 days p.i. Four animals were mock-inoculated and used as uninfected controls. Assigned veterinarians and trained technicians monitored the animals twice daily for signs of distress, including diarrhea, weight loss, and opportunistic infections. Macaques were housed in facilities that are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC), and fed a balanced commercial macaque chow (Purina Mills). Throughout the experiments, animals were housed in cages providing 6 square feet of space with visual and auditory contact of conspecifics, and received environmental enrichment, such as manipulanda and novel foodstuffs. Before euthanasia, all macaques were perfused with sterile saline solution. Organs, including spleen and mesenteric lymph node (MLN), were harvested, sectioned, and frozen at -80C. Blood was collected for the isolation of peripheral blood mononuclear cells (PBMC) using a Percoll protocol (GE Healthcare Life Sciences, Pittsburgh, USA), and cell pellets were frozen and stored at -80C. All animal studies were approved by the Johns Hopkins University Institutional Care and Use Committee, and all procedures followed the guidelines of the Weatherall Report, the USDA Animal Welfare Act, and the Guide for the Care and Use of Laboratory Animals.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from frozen spleen, MLN, and PBMCs using the RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol. Samples were eluted in 60 µL of RNase-free water and frozen at -80oC until time to be analyzed.
Label
Alexa 488, Alexa 594, Alexa 647, Cy3
Label protocol
The nCounter Macaque Inflammatory Genes GAMA CodeSet (NanoString Technologies, Seattle, WA, USA) was used. Samples were labeled according to NanoString's protocol.
Hybridization protocol
Samples were hybridized according to NanoString's protocol.
Scan protocol
Samples were scanned according to NanoString's protocol.
