Analysis of RAPA effects on apoptosis in DCs. RAPA inhibits apoptosis of LPS-stimulated DCs in an FKBP12-dependent manner but does not affect DC apoptosis when cells are cultured for an extended time in the absence of any stimuli (LPS, cytokines). BM-derived DCs were generated with GM-CSF plus IL-4 and washed, and equal numbers of cells were stimulated on day 7 with LPS only or cultured without any stimuli and analyzed after staining of phosphatidylserine translocation with FITC-annexin V in combination with the vital dye 7-AAD. (A) RAPA does not affect apoptosis and death when DCs are cultured for an extended time in the absence of any stimuli. (B-C) RAPA inhibits DC apoptosis and death in a dose-and time-dependent manner when DCs are stimulated with LPS. Competition for RAPA's intracellular receptor FKBP12 by a molar excess of FK506 blocks the inhibitory effect of RAPA on DC apoptosis and death. Doses indicate RAPA, unless stated otherwise. Cells were gated on CD11c. The incidence of cells in each quadrant is indicated. Apoptotic cells are stained annexin V⁺/7-AAD^– and dead cells (late apoptotic or necrotic) are stained annexin V⁺/7-AAD⁺. Results show representative data from 3 (A) and 4 (B-C) experiments. Similar results were obtained with DCs generated in GM-CSF only.