Facs canto

5 × 10⁵ CD4⁺ T-cells were stimulated in 24-well flat bottom plates with either phorbol-12-myristate-13-acetate (PMA; 10⁻⁷ M; Sigma Aldrich) for 30 minutes or anti-CD3/anti-CD28 coated microbeads (2.5 × 10⁵) for 24 hours in the presence of 10 mg/L AZM, 100 nM RAPA or in antibiotic-free medium. Subsequently, cells were harvested and incubated for ten minutes in Fixation Buffer I (BD Phosflow, BD Biosciences) at 37°C. After washing in PBS + 0.5% BSA + 0.05% NaN3 cells were resuspended in pre-chilled (−20°C) Permeabilization Buffer III (BD Phosflow, BD Biosciences) and kept on ice for 30 minutes followed by washing the cells twice in PBS + 0.5% BSA + 0.05% NaN₃. Then, cells were incubated with one of the following antibodies: 20 μL of a p-ERK (T202/Y204; Pacific Blue conjugated; clone 20A), p-p38 (T180/Y182; PE conjugated; clone 36/p38) or p-S6RP (S240; Alexa Fluor 647 conjugated; clone N4-41; all BD Phosflow, BD Biosciences) specific monoclonal antibodies or isotype matched control antibodies for one hour. After washing in PBS + 0.5% BSA + 0.05% NaN₃ cells were cells were analyzed on a FACS Canto flow cytometer using FlowJo software (TreeStar, Ashland, OR).

Mitochondrial membrane potential was examined using The MitoProbe™ DiIC1(5) Assay Kit (Molecular Probe, USA). C2C12 myoblast cells was pre-incubated with either GT (100 ng/mL) in the presence or absence of TNFα (20 ng/mL) for 24 h. Then, cells were stained with 5 μl of 10 μM DiIC1(5) for 15 min at 37°C followed by one wash in PBS buffer. Cells were pelleted by centrifugation at 1000 rpm for 5 min and resuspended in 500 μl of PBS. Fluorescence was analyzed using a FACS Canto and data were analyzed with FlowJo software (Tree Star Inc., USA). The approximate excitation and emission peaks of DiIC1(5) are 638 nm and 658 nm, respectively.

Bronchoalveolar lavage was performed 24h after the last allergen challenge, by intratracheally injecting and aspirating 0.8 ml saline twice. After its collection the BALF was centrifuged for 5 min at 1500 rpm. The cell pellets were resuspended in 1 ml PBS and an aliquot was stained with trypan blue solution and cells were counted using a Neubauer chamber. Eosinophils and neutrophils were detected by fluorescence-activated cell sorting (FACS) analysis. The cell surface staining was performed with antibodies against CD3, GR-1 (BD Bioscience, Heidelberg, Germany), anti-CCR3 (Miltenyi Biotech, Bergisch Gladbach, Germany) and CD45R (eBioscience, Frankfurt, Germany) for 30 min at 4°C. The samples were analyzed by using a FACS-Canto and FlowJo (Treestar Inc).