Carboxyfluorescein succinimidyl ester (cfse)

Adoptive transfer experiments to assess in vivo rejection were performed^{134 (link)}. Splenocytes were isolated from mice lacking expression of murine MHC class I, i.e., H2K^b–H2D^b– (referred to as KO cells), WT mice (which were H2K^b+H2D^b+) or HLA-C*05-expressing mice, which had been bred to mice lacking expression of murine MHC class I molecules (i.e., HLA-C*05⁺ H2K^b-H2D^b-, referred to as HLA-C*05⁺ KO cells). Cells were labeled with differential concentrations of CFSE (Sigma-Aldrich) to allow for cellular discrimination as follows: KO cells (0.5 μM of CFSE), WT cells (5 μM CFSE) and HLA-C*05⁺ KO (5 μM CFSE). Cells were mixed and ~5 × 10⁶ cells per population were injected intravenously into either WT or KIR2DL1-expressing recipient mice. Splenocytes were isolated from recipient mice after 20 h and cells were analyzed by flow cytometry using antibodies for HLA-C (B1.23.2) and H2K^b (AF6-88.5.5.3). A small sample of the injection mix was kept and analyzed by flow cytometry for reference. Survival of both HLA-C*05⁺ KO and KO cells was calculated relative to the WT cells in each experiment using the injection mix as reference. Relative survival of HLA-C*05⁺ KO compared to survival of KO cells was plotted.

Functional regulatory capacity of DM-DCs was assessed by co-culturing DCs with allogeneic CD4+ T cells, which were isolated from peripheral blood using the RosetteSep CD4+ human T cell enrichment cocktail (Stemcell Technologies, Vancouver, Canada) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Sigma-Aldrich) at a concentration of 5 uM. On day 5 after harvest, 10058-F4-treated and untreated DM-DCs were seeded in a 96-well U-bottom plates and co-cultured with CFSE-labeled CD4+ T cells at a 1:2 (DC:T cell) ratio in RPMI medium supplemented with 10% heat inactivated fetal calf serum, at 37°C and 5% CO₂ for 6 days, after that, CFSE dilution was analyzed by flow cytometry as a measure of proliferation, in addition to IFNγ and TNFα production.

All target cells were stained with the green fluorescent cytosolic cell dye carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific, St. Laurent, QC, Canada) to distinguish them from PBMCs or NK effector cells. CFSE staining was performed per the manufacturer’s instructions and as previously described (55 (link)).
For binding experiments and ADCC assays using two target cells combined (i.e., rgp120-coated CEM cells or infected CEM cells 4 days postinfection (p.i.) with HIV combined with uncoated or uninfected CEM cells), CFSE⁺ cells were also stained with PKH26 red fluorescent membrane cell dye (PKH26 red fluorescent cell linker kit; Sigma-Aldrich) to distinguish them from CFSE⁺ PKH26⁻ uncoated or uninfected CEM control cells, used as an internal control for nonspecific binding and killing. PKH26 staining was performed as previously described (55 (link)).