Cmtpx

HSV-TK expressing MSCs (MSC_HSV-TK) were incubated with or without 100 μM gap junction inhibitor Carbenoxolone (Sigma) for 16-20h before cell detachment. Cells were harvested, washed in PBS and stained with 5 μM CMTPX (gap junction impermeable; Molecular Probes) and 1 μM Calcein AM (gap junction permeable; Molecular Probes) for 20 min at 37°C. After washing, MSCs were mixed with tumor cells at a ratio of 1:1 and were plated at a density of 1x10⁵ cells/24-well. To assess cell contact independent dye transfer, 5x10⁴ double stained MSC were seeded in the top well of a 96 transwell-plate, 5x10⁴ non-labeled tumor cells were placed in the bottom well. Cells were analyzed by flow cytometry directly after mixing and after 4h of (co-) cultivation to evaluate dye transfer of Calcein AM to unstained tumor cells.

Blood from infected B6 mice was resuspended in 1 ml PBS, pipetted over 5 ml of 74% Percoll (GE Healthcare, USA) and centrifuged (2500 x g, acceleration/break 5/0) for 30 min at room temperature (RT). The top cell layers were collected and washed with complete RPMI 1640 medium (supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol, 2 mM L-glutamine and 1 mM sodium pyruvate; Life Technologies, USA). Purified iRBCs (>95% purity) were stained with CTV or CMTPX, following the manufacturer’s instructions (Life Technologies).

Inflammation was induced in the ears of C57BL/6 mice by intradermal injection of murine TNFα (10 μg (R and D Systems) and IL-1β (1 μg (R and D Systems) in 20 μl PBS. Eighteen hours after injection, 1 × 10⁶ human T cells were injected into the left ventricle, and mice were euthanized 8 min later. Ears were removed, ear sheets were split and cartilage and fat were scraped off. The sheets were then immediately fixed in cold acetone for 20 min for tissue staining or treated in DMEM (Invitrogen, Carlsbad, CA) containing 1 mg/ml DNase I (Sigma-Aldrich St. Louis, MO) and 250 μg/ml Liberase TM (Roche Custombiotech, Indianapolis, IN) for 50 min at 37°C to obtain cell suspensions. Cells were then filtered through a 70 μm nylon mesh and washed prior to counting using the flow cytometer. In some cases, the T cells were labeled with either CMTPX (Life Technologies) or CFSE (Life Technologies) prior to injection.

Antibody-free mature Pc-iRBCs (>95% late trophozoites/schizonts) were obtained from the blood of infected RAGKO mice. Blood samples (500 μl) were resuspended in 1 ml PBS, pipetted over 5 ml of 74% Percoll (GE Healthcare) and centrifuged (2,500 x g, acceleration/break 5/0) for 30 min at RT. The top cell layers were collected and washed with supplemented RPMI 1640 medium. Purified mature Pc-iRBCs (>95% purity) were stained with 5 μM SYTO 16 (Invitrogen) or CMTPX (Life Technologies) following the manufacturer’s instructions.

To demonstrate that virus transmission from infected MDM to VERO E6 cells was mediated by cell membrane fusion with cytoplasmic material exchange, two vital fluorescent dyes were used: 5-chloromethylfluorescein diacetate (CMFDA, Invitrogen, Waltham, MA, USA) and 4-({[4-(chloromethyl)phenyl]carbonyl}amino)-2-(1,2,2,4,8,10,10,11-octamethyl-10,11-dihydro-2H-pyrano[3,2-g:5,6-g’]diquinolin-1-ium-6-yl)benzoate (CMTPX, Invitrogen). Each dye was used as a 5 mM stock solution in dimethylsulfoxide (Merck, Kenilworth, NJ, USA). Overall, 2 × 10⁵ infected and uninfected MDM were stained with 1 mM CMTPX (Cell Tracker Red), whereas 3 × 10⁵ VERO E6 were stained with 1 mM CMFDA (Cell Tracker Green) according to a reported procedure [16 (link)] and seeded in a 24-well microplate for 24 h. The day after, infected or uninfected red MDM were inoculated onto uninfected green VERO E6 and incubated for 24 h at 33 °C, 5% CO₂. The microscopic observation of VERO E6 cells and their dual fluorescing fusion products (orange/yellow merging color) was achieved by using a fluorescent microscope (model DM RBE; Leica, Wetzlar, Germany) with filter G/R DM 513803 (Leitz) designed for simultaneous excitation by 490 +/− 20 and 575 +/− 30 nm fluorescent light.