Celltrace far red reagent

Cultured MSCs labeled using the CellTrace Violet Cell Proliferation Kit (Invitrogen) were infected with vMyx-EGFP/tdTr (MOI = 10) for 1 h (5% CO₂, 37°C). The vMyx-EGFP/tdTr tandem system allows expression of EGFP at both early and late infection stages (early/late promoter), while tdTr is expressed only at the late infection stage (poxvirus p11 late promoter).³⁷ (link) Unbound virus was washed away and 50 μg/mL of Ara-C solution (Ara-C is an inhibitor of poxviral DNA replication and late gene expression of MYXV) was added to evaluate the infection. After 24 h, B16-F10 melanoma cultures were stained with CellTrace Far Red reagent (Invitrogen), and, following trypsinization, melanoma cells were added to the Ara-C-treated or untreated MSC cultures (1:1 cell-to-cell ratio). The co-culture was incubated further (5% CO₂, 37°C) up to 48 h. Infection was evaluated using fluorescence microscopy (Leica) and flow cytometry (BD FACSCanto).

GFP-Egr2–transduced cells were labeled with CellTrace Far-Red reagent (Invitrogen). The cells were stimulated for 72 h with 5 µg/ml anti-CD3 and 2 µg/ml anti-CD28 before analysis by flow cytometry.
Purified CD8 cells (5 × 10⁴) were incubated with OVA-VV_WR–infected LB27.4 cells at 1:1 or 1:5 ratios in 96-well plates in triplicate for 48 h. A total of 1 µCi of [³H]TdR was added for the last 8 h of culture, and the cells were then harvested and subjected to scintillation counting to measure [³H]TdR incorporation.