Flow cytometric analysis and intracellular staining
For intracellular staining total lung cells from Balb/c wild-type mice
were incubated over night with anti-CD3 antiCD28 antibodies and then
stimulated for 4h with PMA/ Ionomicin and a protein transport inhibitor
according to the manufacturer‘s protocol. Cells were harvested, washed
and stained with anti-CD4, anti-CD8 and anti-CD25 antibodies for 30 min
at 4°C and then stained with anti-Foxp3, anti IFN-gamma and anti-IL10
antibodies in staining buffer (BD Biosciences, Heidelberg) for 35 min at
4° C. Afterwards cells were washed again and resuspended in stain
buffer. The following anti-mouse antibodies were used: CD4 FITC (BD
Biosciences), CD4 PerCP (BD), CD25 PerCP (BD), Foxp3 APC (Miltenyi
Biotec, Bergisch Gladbach, Germany).
Lung cell culture
WT Bl6/C57 and ST2-/- total lung cells were incubated in the presence of
Vitamin D3, OVA and with or without RV infection at 37°C. After 24h
cells were harvested, washed and stained with anti-CD4 and anti PD-1
antibodies in staining buffer (BD Biosciences, Heidelberg) for 30 min at
4°C. Marked cells were acquired by using FACS-Calibur (BD Biosciences,
Heidelberg) and analyzed with FlowJo.