FCM detection of lipid bodies (LBs) in control C57BL/6 DLs and C57BL/6 DLs hosting DsRed2 L. am amastigotes.

Panel A. Representative analysis of the fluorescence of BODIPY 493/503 in MHC II⁺ DLs. A1, A2. Control C57BL/6 DL cultures –i.e. not exposed to L. am amastigotes- were incubated or not at 34°C with 200 µM oleate for 21 hours. A3, A4. DsRed2-L. am were added to DL cultures at a ratio of 5 DsRed2-L. am per DL (+L. am). Oleic acid (200 µM) was added 3 hours post inoculation, (A4: +Oleate) or not (A3: −Oleate) for a further 21 hours at 34°C. Control and L. am-loaded DLs were then detached. LBs were stained with BODIPY 493/503 2 µg/ml in PBS for 30 minutes at 34°C. The cells were then incubated with anti MHC II- PE-Cy5 mAb to analyze only the MHC class II-positive DLs. FCM histograms show BODIPY 493/503 fluorescence for Ctrl (A1, A2: white histograms) and DsRed2 L. am-hosting DLs (A3, A4) (DsRed2⁺, black histograms) and amastigotes-free DLs (DsRed2⁻, grey histograms). Mean fluorescence intensity was indicated for each histogram. Panel B: Statistical analysis of the BODIPY 493/503 MFI values. Mean BODIPY 493/503 fluorescence by MHC II⁺ DLs was determined for n = 7 independent experiments. MFI in L. am-loaded cultures is shown for DsRed2⁺ and DsRed2⁻ DLs as black and grey bars, respectively. Statistical analyses were performed by the Mann Whitney test.