Celltrace far red ctfr

In vivo cellular cytotoxicity assay were adapted from Durward et al. (30 (link)). In brief, splenocytes were harvested and single cell suspensions prepared. Splenocytes were incubated with the MHC class I restricted influenza HA (Cal07) peptide IYSTVASSL, the NP peptide (RLIQNSLTIERMVLS), or a negative control peptide, at a density of 5x10⁷ cells/mL for 1 h at 4°C followed by 30 min incubation at 37°C. Peptide‐loaded cells were washed twice in PBS and subsequently stained with 5 μM CellTrace Violet (CTV) (C34557, Life Technologies) (HA peptide loaded cells), or 1 μM CellTrace Far Red (CTFR) (C34564, Life Technologies) (NP peptide loaded), or double stain (CTV and CTFR) (negative control) at a density of 5x10⁷ cells/mL for 20 min at 37°C. Cells were mixed in equal ratios (1:1:1), and a total of 15x10⁶ cells injected i.v. in a 100µl volume to vaccinated mice. Spleens were harvested 16h later, single cell suspensions prepared, and the presence of peptide loaded cells investigated by flow cytometry. The ratio of CTV to CTV/CTFR or CTFR to CTV/CTFR cells were calculated as % specific lysis = [1 − (average ratio in group with NaCl vaccinated mice/experimental ratio)].

To analyze the formation of syncytia between Jurkat‐S and HepG2 cells, HepG2 cells were detached from the culture plate by trypsinization and resuspended at a concentration of 3 × 10⁶ cells/ml in PBS. Jurkat‐S cells were collected by centrifugation and resuspended in PBS at the same concentration. HepG2 cells were labeled with Cell Trace Far Red (CTFR, Invitrogen) for 5 min at 37°C in PBS; Jurkat‐S were labeled with CFDA‐SE (CFSE, Invitrogen) under the same conditions. Both dyes were used at a final concentration of 5 μM. Labeling was stopped by adding complete medium and the cells washed with medium and finally mixed at different ratios in complete RPMI medium + 5% FBS and plated overnight at 37°C. Formation of syncytia was analyzed by flow cytometry by calculating the percentage of cells double positive for CFSE and CTFR. Syncytia formation was confirmed by confocal microscopy. Syncytia was selected by FACS sorting and allowed to bind to poly‐l‐lysine‐coated glass for 30 min and fixed for 10 min at room temperature with 4% paraformaldehyde. Cell nuclei were stained with 1 µg/ml DAPI in PBS for 5 min, and finally, samples were mounted with Prolong Antifade (Molecular probes). Samples were observed by LSM 710 laser scanning confocal microscope. Image processing was performed using Zen software.