D in a ventilated room with all appropriate hygiene and feeding conditions throughout the experiments. Housing, inoculation, data collection, and euthanasia procedures complied with National Institutes of Health guidelines, and the experimental protocol was approved by the Bioethics Committee of the University of Liege. Mice were monitored twice daily and in the ` case of a weight loss exceeding 30 of the body weight or clear signs of animal suffering, mice were euthanized by cervical dislocation and integrated in the mortality curve data.Quantification of leukocyte infiltration by flow cytometry analysisFlow-cytometry data acquisition was performed on a dual-laser FACSCalibur flow cytometer running CELLQuest software (BD Biosciences, Erembodegem, Belgium). WinMDI software was used for data analysis. Cells were stained with mAbs directed against F4/80 (FITC), CD11b (PerCp-Cy 5.5), CD4 (PerCP), CD11c (APC), I-A/I-E (PE), CD19 (PE), Ly-6G and Ly-6C (PerCp-Cy 5.5), CD8 (FITC), CD3 (APC), and isotype 18325633 controls (all from BD Biosciences except F4/80-FITC from AbD Serotec).Quantification of cytokine levels in BALFs of P2Y2+/+ and P2Y22/2 miceCytokines such as KC, MIP-2, MIP-1a, MCP-1, IL-12p40, IFN-c, TNF-a, IL-6, IFN-b, IL-17, MIP-3a, IP-10 were measured in P2Y2+/+ and P2Y22/2 BALFs using ELISA kits from BD Biosciences and R D Systems (Abingdon, U.K.), following the manufacturer’s instructions. BRAK was measured by RT-qPCR using the following primers set: 59-GAT GAA GCG TTT GGT GCT CT-39 and 59-AGT ACC CAC ACT GCG AGG AG-39, with Power SYBR Green PCR Master Mix (Applied Biosystem). Reactions were run on a 7500 Fast Real-Time PCR System (Applied Title Loaded From File Biosystems). The cycling conditions were 10 min for polymerase activation at 95uC and 40 cycles at 95uC for 15 s and 60uC for 60 s. Mean 6 SD values were obtained for each gene using qBase software. Each assay was performed in duplicate.AnimalsP2Y2R knockout (P2Y22/2) mice were provided as breeder pairs (on a B6D2 genetic background) by Dr. B.H. Koller (University of North Carolina, Chapel Hill, NC). The B6D2 P2Y22/2 mice were then crossed with the SV129 mouse strain by Dr. J. Leipziger (Institute of Physiology and Biophysics, University of Aarhus, Aarhus, Denmark), generating B6D2/SV129 P2Y2+/+ and B6D2/SV129 P2Y22/2 littermates. Mice were then backcrossed onto C57Bl6 for .10 generations. The experiments were conducted with specific pathogen-free 8-week-old Title Loaded From File female miceViral TitrationAt day 8 and day 10 post-infection, mice were euthanized to quantify lung virus titers by quantitative polymerase chain reaction (qPCR) as previously described [21]. The lungs were homogenized in ice-cold BSA 1 in PBS, and clarified (1000 g for 10 min). Viral RNA was extracted using Nucleospin RNA Virus columns according to the user manual (Macherey Nagel). Homogenates were treated with Fermentas DNase I and an aliquot of each RNA extract (100 ng RNA) was then reverse-transcribed using commercial high capacity cDNA reverse transcription kit (Invitrogen), and PCR was conducted using the following PVM SH gene primers set: 59-GCC GTC ATC AAC ACA GTG TGT-39 and 59-GCC TGA TGT GGC AGT GCT-39, with SYBR green PCR Master Mix (Applied Biosystem). SDHA was selected as control gene after analysis for its stability in our system. Reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems). The cycling conditions were 10 min for polymerase activation at 95uC and 40 cycles at 95uC for 15 s and 60uC for 60 s. Mean 6 SD.D in a ventilated room with all appropriate hygiene and feeding conditions throughout the experiments. Housing, inoculation, data collection, and euthanasia procedures complied with National Institutes of Health guidelines, and the experimental protocol was approved by the Bioethics Committee of the University of Liege. Mice were monitored twice daily and in the ` case of a weight loss exceeding 30 of the body weight or clear signs of animal suffering, mice were euthanized by cervical dislocation and integrated in the mortality curve data.Quantification of leukocyte infiltration by flow cytometry analysisFlow-cytometry data acquisition was performed on a dual-laser FACSCalibur flow cytometer running CELLQuest software (BD Biosciences, Erembodegem, Belgium). WinMDI software was used for data analysis. Cells were stained with mAbs directed against F4/80 (FITC), CD11b (PerCp-Cy 5.5), CD4 (PerCP), CD11c (APC), I-A/I-E (PE), CD19 (PE), Ly-6G and Ly-6C (PerCp-Cy 5.5), CD8 (FITC), CD3 (APC), and isotype 18325633 controls (all from BD Biosciences except F4/80-FITC from AbD Serotec).Quantification of cytokine levels in BALFs of P2Y2+/+ and P2Y22/2 miceCytokines such as KC, MIP-2, MIP-1a, MCP-1, IL-12p40, IFN-c, TNF-a, IL-6, IFN-b, IL-17, MIP-3a, IP-10 were measured in P2Y2+/+ and P2Y22/2 BALFs using ELISA kits from BD Biosciences and R D Systems (Abingdon, U.K.), following the manufacturer’s instructions. BRAK was measured by RT-qPCR using the following primers set: 59-GAT GAA GCG TTT GGT GCT CT-39 and 59-AGT ACC CAC ACT GCG AGG AG-39, with Power SYBR Green PCR Master Mix (Applied Biosystem). Reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems). The cycling conditions were 10 min for polymerase activation at 95uC and 40 cycles at 95uC for 15 s and 60uC for 60 s. Mean 6 SD values were obtained for each gene using qBase software. Each assay was performed in duplicate.AnimalsP2Y2R knockout (P2Y22/2) mice were provided as breeder pairs (on a B6D2 genetic background) by Dr. B.H. Koller (University of North Carolina, Chapel Hill, NC). The B6D2 P2Y22/2 mice were then crossed with the SV129 mouse strain by Dr. J. Leipziger (Institute of Physiology and Biophysics, University of Aarhus, Aarhus, Denmark), generating B6D2/SV129 P2Y2+/+ and B6D2/SV129 P2Y22/2 littermates. Mice were then backcrossed onto C57Bl6 for .10 generations. The experiments were conducted with specific pathogen-free 8-week-old female miceViral TitrationAt day 8 and day 10 post-infection, mice were euthanized to quantify lung virus titers by quantitative polymerase chain reaction (qPCR) as previously described [21]. The lungs were homogenized in ice-cold BSA 1 in PBS, and clarified (1000 g for 10 min). Viral RNA was extracted using Nucleospin RNA Virus columns according to the user manual (Macherey Nagel). Homogenates were treated with Fermentas DNase I and an aliquot of each RNA extract (100 ng RNA) was then reverse-transcribed using commercial high capacity cDNA reverse transcription kit (Invitrogen), and PCR was conducted using the following PVM SH gene primers set: 59-GCC GTC ATC AAC ACA GTG TGT-39 and 59-GCC TGA TGT GGC AGT GCT-39, with SYBR green PCR Master Mix (Applied Biosystem). SDHA was selected as control gene after analysis for its stability in our system. Reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems). The cycling conditions were 10 min for polymerase activation at 95uC and 40 cycles at 95uC for 15 s and 60uC for 60 s. Mean 6 SD.
