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HeLa cells transfected with rat Cx43 containing a carboxyl terminal (His)6 epitope, with chicken Cx45, or with both connexins have been described and characterized previously [Cx43: Accession X06656M19317; Cx45 Accession NM 205503(Martinez et al., 2002 (link))]. HeLaCx43 or HeLaCx45 cells were co-cultured at a ratio of 1:1 with the co-transfected HeLaCx43/Cx45 cells allowing the formation of mono-heteromeric channels with Cx43 (MhetCx43) and Cx45 (MhetCx45) homomeric connexons, respectively. In this manuscript, we will refer to rat Cx43(His)6 and chicken Cx45 as Cx43 and Cx45. Rat (Rattus norvergicus) Cx43 and chicken (Gallus gallus) Cx45 present 83% identity. The main differences are located at the intracellular loop and at the CT domain. The first extracellular loops have 100% identity. The second extracellular loops have 85% identity, with no differences in critical residues relevant for stabilization of docking connexons. For discrimination under epifluorescence microscopy, co-transfected cells expressing the heteromeric connexons were stained with 100 μM DiI Red (Molecular Probes Inc. D-282), while cells that expressed Cx45 or Cx43 were loaded with 50 μM Cell Tracker Green (Molecular Probes Inc. C-2925), both for 50 min at 37°C. To identify injected cells after fixation, cells were plated on coverslips with a central etched grid (Cell-Locator, CA).

Rhodamine Lens Culinaris Agglutinin and VectaShield with DAPI were obtained from Vector Laboratories (Burlingame, CA). Fluospheres, Quant-iT pico green, Cell Tracker green and Texas Red-conjugated bovine serum albumin (BSA) were obtained from Molecular Probes (Eugene, OR). Fucoidin was obtained from Sigma Chemical Company (St. Louis, MO).

Macrophages (∼2.5×10⁶ cells) were incubated the day before the experiment for 1 h at 37°C with stationary phase Leishmania promastigotes (ratio 20:1, parasites/macrophages). Non-internalized parasites were then removed by excessive washing, fresh medium was added and the incubation was continued for 1–3 days. At the end of the incubation period, the macrophages were washed twice with PBS and lysed with 0.01% (v/v) SDS in PBS (30 min, RT), to release internalized parasites. Broken macrophages were removed by centrifugation (250 g, 5 min). The parasites in the supernatant were collected by centrifugation (2000 g, 10 min) and labelled [30 min, room temperature (RT)] with Cell Tracker Green (CMFDA, Molecular Probes®; 5 μM in PBS). Finally, the cells were washed with PBS and analysed by FACS (FACS Calibur, BD Biosciences). To define the population of apoptotic/necrotic cells incorporating CMFDA, the parasites were treated with 4 mM H₂O₂ for 12 h before labelling [39 (link)]. On the basis of this analysis the histogram gates for live or apoptotic/necrotic cell population were set as M3 and M2 respectively.

Healthy donor PBMCs were activated with HSV^GM-CSF overnight at the indicated concentrations, and their ability to kill melanoma cell targets (with or without VPA treatment) stained with Cell Tracker Green (Molecular Probes) was determined using standard 5-h co-culture. Co-cultures were washed and stained for viability using a live-dead fixable dead cell stain (Thermo Fisher Scientific) before analysis using an Attune flow cytometer.