Diva program

Cell suspensions from the lamina propria were prepared as described previously^{36 (link)}. All cells were first pre-incubated with anti-mouse CD16/CD32 for blockade of Fc γ receptors, then were washed and incubated for 40 min with the appropriate monoclonal antibody conjugates in a total volume of 200 μl PBS containing 2 mM EDTA and 2% (vol/vol) bovine serum. DAPI (Invitrogen) was used to distinguish live cells from dead cells during cell analysis and sorting. For detection of intracellular Foxp3, a Foxp3 Staining Buffer Set (eBioscience) was used for fixation and permeabilization of the cells. Stained cells were analyzed on a LSRII machine using Diva program (BD Bioscience) or purified with a MoFlo Astrios cell sorter (DakoCytomation). Cells were >98% pure after sorting. Data were analyzed with FlowJo software (TreeStar).

Propidium iodide (BD Bioscience) and the fluorescein isothiocyanate-conjugated (FITC) anti-human Annexin V Apoptosis Detection Kit I (BD Pharmingen) were used to characterize cells. Labeled cells (1 × 10⁶) were analyzed using the BD FACS Aria II Cell Sorter System (BD Biosciences), followed by data analysis using the Diva program (BD Biosciences).

Cell populations were treated with erufosine, washed with PBS and fixed in 70% ethanol-PBS solution for 1 h on ice. Fixed cells were washed twice with ice cold PBS, treated with RNAse A for 30 min at 37°C and stained with propidium iodide 15 min prior to analysis. Cellular DNA content was determined by flow cytometry using the DIVA Program (BD) and ModFit LT software.