Facscalibur flow cytometer

Mouse tissue cell suspensions, at 2×10⁸ cells/ml, were incubated with purified anti-Fc receptor blocking antibody [anti-CD16/CD32] before addition of the specific antibodies. Cell surface markers were stained using a combination of fluorescein isothiocyanate [FITC]-, phycoerythrin [PE]-, PE-Cy7- and allophycocyanin [APC]-conjugated monoclonal antibodies directed against: T-cells CD3 [clone 17A2], CD4 [GK1.5], CD8 [53-6.7], B-cells CD19 [1D3], Neutrophils Gr-1 [RB6-8C5], Macrophages F4/80 [BM8]. Where appropriate, CD45 [30-F11] was used to identify haematopoietic cells. In each experiment the appropriate isotype control monoclonal antibodies and single conjugate controls were also included. All antibodies were purchased from eBioscience. Samples were analysed using a Becton Dickinson FACScalibur flow cytometer running CellQuest acquisition and analysed using FlowJo software [version 8.8.3, Tree Star].

Normal and GFP-expressing limb bud-polarizing regions were dissected in PBS under a LeicaMZ16F UV microscope using fine surgical scissors, pooled from replicate experiments (between 10 and 12), and digested into single-cell suspensions with trypsin (0.5%, Gibco) for 30 min at room temperature. Cells were washed twice in PBS, fixed in 70% ethanol overnight, washed twice in PBS and re-suspended in PBS containing 0.1% Triton X-100, 50 μg ml⁻¹ of propidium iodide and 50 μg ml⁻¹ of RNase A (Sigma). After incubation at room temperature for 20 min, cells were analysed for cell cycle distribution with a FACSCalibur flow cytometer and FlowJo software (Tree star Inc). Cells with a DNA content between 2N and 4N were designated as being in the G1, S, or G2/M phase and expressed as a percentage of the total number of cells present (6,000–10,000). Statistical significance of cell cycle phase values was determined by Pearson’s χ² tests to obtain two-tailed P-values (significantly different being a P-value of <0.05).

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep^TM (Axis‐Shield, Heidelberg, Germany) for processing and analysis by flow cytometry, reverse transcriptase–polymerase chain reaction (RT‐PCR) and in vitro Treg suppression assays. The following antibodies were used for sorting of PBMCs using a FACSAria IIu cell sorter (BD Biosciences, San Jose, CA, U.S.A.): fluorescein–isothiocyanate (FITC)‐conjugated antihuman CD4 (clone RPA‐T4), PE‐Cy7‐conjugated antihuman CD25 (M‐A251), phycoerythrin (PE)‐conjugated antihuman CD127 (clone hIL‐7R‐M21), all from eBioscience (Vienna, Austria). We used PE‐conjugated antihuman CD127 (clone hIL‐7R‐M21), FITC‐conjugated antihuman CD25 (clone 2A3), peridinin chlorophyll protein complex (PerCP)‐conjugated antihuman CD4 (clone SK3) (BD Pharmingen, San Diego, CA, U.S.A.) and allophycocyanin (APC)‐conjugated antihuman FoxP3 (clone PCH101) from eBioscience for quantification of human PBMCs. Data was acquired on a FACSCalibur flow cytometer and analysed with FLOWJO software (version 7.6.5; TreeStar Inc., Ashland, OR, U.S.A.). All plots were pregated on CD4⁺ lymphocytes. The increase in percentage of Tregs as a proportion of either the CD4 subpopulation or the entire lymphocyte subpopulation was calculated by dividing TP2 by TP1.

For intracellular staining, splenocytes were incubated with brefeldin A, an intracellular protein transport inhibitor (10 μg/mL), in RPMI medium (Gibco BRL, Gaithersburg, MD, USA) for 2 h at 37 °C. The cells were stained for cell surface markers, fixed with 1% paraformaldehyde, washed once with cold FACS buffer, and permeabilized with 0.5% saponin. The permeabilized cells were then stained for an additional 30 min at room temperature with the indicated mAbs (PE-conjugated anti-IFN-γ, anti-IL-4, anti-IL-5, anti-IL-17, or PE-conjugated isotype control rat IgG) [36 (link)]. More than 5000 cells per sample were acquired using a FACSCalibur flow cytometer, and the data were analyzed using the FlowJo software package (version 8.7; Tree Star, Ashland, OR, USA).

The levels of IFN-γ, TNF-α, interlukin-6 (IL-6), IL-10, IL-12p70, and monocyte chemoattractant protein-1 (MCP-1) in bronchoalveolar lavage (BAL) fluid or lung tissue were analyzed using a BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions with the exception that a total of 2 μl of each capture bead was used in 50 μl of BAL sample and the PE-detection reagent was diluted 1 in 5. Samples were analyzed using a Becton Dickinson FACSCalibur flow cytometer and data analyzed using the FlowJo software package (Tree Star, Inc., Ashland, OR, USA).