Celltracker orange cmtmr

HIV-1 proviral constructs were transduced into Jurkat cells (donor cells) using Amaxa nucleofection as previously described (Amaxa Biosystems). In brief, 5 μg of endotoxin-free HIV-1 proviral plasmids was nucleofected into 6 × 10⁶ Jurkat cells using Cell Line Nucleofector kit V, program S-18. Twenty hours after nucleofection, viable Jurkat cells were purified by centrifugation on a Ficoll-Hypaque density gradient. Twenty-four hours after nucleofection, cells were washed with complete buffer and recovered at 37 °C for co-culture. Unactivated primary CD4⁺ T cells (target cells) were resuspended in serum-free RPMI 1640 and stained with 1 μM Cell-Tracker Orange CMTMR (5-and-6-(((4chloromethyl)benzoyl)amino)tetramethylrhodamine); Molecular Probes) for 45 min at 37 °C, washed, and cultured overnight in complete RPMI medium containing 50 U/ml IL-2. Donor and target cells were mixed at a ratio of approximately 1:1 and cocultured at 37 °C for 3 h before they were treated with trypsin and fixed. Where inhibitor Leu3a, an HIV-blocking anti-CD4 antibody (BD Biosciences) was used, donor and target cells were preincubated separately with equal volumes of inhibitor for 30 min at 37 °C before mixing.

Cell mixture was performed as described previously [5 (link),43 (link)]. Briefly, the cell type different from β₂ YFP transfected CHO fibroblast (CHO wt or MDCK β₂ –His₆) was incubated with CellTracker Orange CMTMR (Molecular Probes, Eugene, OR, USA) for 1 h on 37 °C at a final concentration of 6.0 μM. Cells were then washed three times with PBS solution, re-incubated for 1 h in DMEM supplemented with 10% FCS or FBS. The day after, cells were harvested by trypsin and the suspension was mixed with CHO β₂ YFP cells suspension, in different proportions, as indicated in each case, to be co-cultured on glass coverslips and processed the day after for immunofluorescence assay. The same procedure was implemented when the cell mixture was analyzed for cell aggregation except for when the mixing ratio was 3:1, 1:1, and 1:3. For Co-AP assay, MDCK β₂-His₆ cells were co-cultured with CHO β₂ YFP cells or with MDCK β₂ YFP cells in a 3:1 ratio without using a CellTracker.

The breast cancer cell line, SKBR-3(ATCCHTB-30) and MCF-7(ATCCHTB-22) cells, were cultured in Dulbecco’s Modified Eagle Medium (Sigma Aldrich, Zwijndrecht, NL), supplemented with 10% Fetal Bovine Serum (Sigma Aldrich), 2 mM L-Glutamin (Sigma Aldrich), 1% Penicillin-Streptomycin (Sigma Aldrich) at 37°C in 5% CO2 atmosphere. Before experimentation, cells were stained with CellTracker Orange CMTMR (Molecular Probes, Breda, NL) at 37°C for 30 min and detached by 0.05% of Trypsin/EDTA (Gibco, Paisly, UK). Thereafter, cells were washed once with the culture medium and re-suspended in PBS solution.

Cancer cell lines and human umbilical vein endothelial cells (HUVECs) were stained by 5 µM (in PBS) 5-chloromethylfluorescein diacetate (CellTracker Green CMFDA, Molecular Probes, UK) and (5 µM) 5-(and-6-)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (CellTracker Orange CMTMR, Molecular Probes, UK), respectively. The probes were diluted from 10 mM stock solution in DMSO into PBS and incubated with the cells at 37 °C, 5% CO₂ for 30 minutes. These probes were designed for relatively long-term tracking, lasting for at least three generations of passages and therefore chosen for this study. The images were taken using a fluorescence microscope (Nikon TE-2000, UK) for analysis. The details for microscope parameters are provided in the Supplementary Data.

The conjugation assay was performed according to a previously described protocol [32 (link)]. NKL cells loaded with CFSE and 221 cells labeled with CellTracker orange CMTMR (Molecular Probes, Waltham, MA, USA) were separately chilled on ice and then mixed at an E: T ratio of 1:1. Cells were spun down at 30× g for 3 min and then incubated at 30 °C for the indicated times. Thereafter, cells were moved to ice, fixed in PBS containing 4% paraformaldehyde, and washed twice with FACS buffer. Conjugates (CFSE+CMTMR+) were detected by flow cytometry.

One hour prior to co-culture, 10,000 of HUVECs and either control and Sdc-1-silenced SUM-149 or MDA-MB-231 cells were maintained in 50 μL Endothelial Basal Medium extract (BEM) (Corning^® Matrigel^® Basement Membrane Matrix Growth Factor Reduced, cat. no. 354230) and then cells were grown in a 96-well plate as follows: HUVECs were maintained in BEM with and without FCS, control, and Sdc-1 siRNA transfected cells, HUVECs with control, and Sdc-1 siRNA transfected cells. The 96-well plate was maintained for 24 h in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were stained with Cell Tracker Green CMFDA (Molecular Probes, Thermo Fisher Scientific, cat. no. C2925, Massachusetts, MA, USA) or Cell Tracker Orange CMTMR (Molecular Probes, Thermo Fisher Scientific, cat. no. C2927) according to the manufacturer’s instructions and monitored using confocal time-lapse microscopy (LSM 880), followed by quantitative analysis of tube formation as described above. For TF pathway inhibition, the aforementioned steps were analogously repeated in absence and presence of 50 ng/mL recombinant human CellExpTM tissue factor pathway inhibitor (TFPI), (Sigma-Aldrich Chemie GmbH, cat. no. SRP6458-10 UG).