Flow Cytometric Analysis of Lamina Propria Cells

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).