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Flow cytometric assessment of PBMCs. Apoptosis was determined using the APO-BrdU kit (Phoenix Flow Systems) according to manufacturer’s instructions. Fluorescently-labeled mAbs identified CD3 T-cells (BD Pharmingen). Cell permeability and extracellular phosphatidyl serine were measured simultaneously by staining MP470 inhibitor with Annexin-V-FITC (BioVision) and 7AAD (BD Pharmingen) according to manufacturer’s instructions. Flow cytometry (25,000–50,000 events/sample) was performed on FACScan (BD Biosciences).
In vitro assessment of cellular uptake and subcellular distribution. Primary human lymphocytes were treated with 5 μM FITC-labeled TAT-BH4 or NLS-BH4 and incubated for 1 h in either RPMI with 10% FBS or sterile-filtered, serum-free PBS, washed twice with PBS, and assessed by flow cytometry. A second identically prepared cohort of cells was treated with Alexa 595-conjugated Wheat Germ Agglutinin (WGA) (Invitrogen) according to manufacturer’s instructions to visualize the plasma membrane. Cells were washed again, resuspended in RPMI with 10% FBS, and added to coverslip-bottomed 35 mm culture dishes (MatTek) for imaging. Images (400×) were taken sequentially to prevent fluorochrome cross-talk. Gain and photomultipliers were the same for all samples. In fluorescence microscopy studies, PBMCs were treated as above with the fluorescently-labeled peptides, omitting the WGA labeling. Cells were deposited on slides for imaging (600×, Nikon Eclipse E600).