7 aminoactinomycin d 7 aad viability staining solution

Before flow cytometry analysis, all cells were washed with PBS and resuspended in autoMACS® running buffer (Miltenyi Biotec, Bergisch Gladbach, Germany). The antibodies and isotype antibodies used for flow cytometry are listed in Supplemental Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/6579463. Dead/live staining was conducted with a 7-amino-actinomycin D (7-AAD) Viability Staining Solution (eBioscience, San Diego, CA, USA). For the assessment of all cells, FcR block was performed by incubation with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 8 min. Multicolour flow cytometry was conducted on a MACSQuant™ analyser (Miltenyi Biotec, Bergisch Glattbach, Germany). FoxP3 intracellular staining was performed after fixation using the FoxP3 staining buffer set (Miltenyi Biotec, Bergisch Glattbach, Germany) according to the manufacturer's protocol. The MACSQuantify 2.1 software (Miltenyi Biotec, Bergisch Glattbach, Germany) was used for data analysis. Positive fluorescence was defined as any event above the background fluorescence in a histogram. Background fluorescence was defined by a cutoff where 99.5% of the background fluorescence events matched to isotype antibody results were marked negative.

Changes in cell viability were assessed by flow cytometry using the Annexin V Apoptosis Detection Kit and the 7-amino-actinomycin D (7-AAD) viability staining solution (eBiosciences). Cells (2–5 × 10⁵) were labelled according to the supplier’s instructions. The percentage of viable cells was measured by using a Coulter EPICS XL flow cytometer with the Expo32 software (Beckman Coulter).