Ivis lumina xrms in vivo

Luciferase gene expression was quantified using BLI (IVIS Lumina XRMS in vivo imaging system, PerkinElmer, Massachusetts, USA) at various imaging time points indicated in Fig. 1. Investigators were not blinded to the group allocation during the bioluminescence imaging. Mice were anaesthetised as described above and 50 µl of 15 mg/ml D-luciferin (Cayman Chemicals, US) was delivered to both nostrils as a bolus over 10 to 20 s. Ten minutes later each animal was imaged following a previously described method [10 (link)]. The resultant photon flux was determined in a region of interest created using the auto contour parameter measurement tool with background correction. After the final imaging time point, all mice were humanely killed with sodium pentobarbital (150–300 mg/kg i.p.) while under anaesthetic. Lungs with the trachea attached were immediately excised and re-imaged ex vivo in a small petri dish containing phosphate-buffered saline [10 (link)], to remove the obstructive effect of body tissues and fur on the detectable bioluminescence.

The TC71 ES line was engineered to express a red-shifted Luciola italica luciferase transgene using RediFect™ Red-FLuc-Puromycin Lentiviral Particles (PerkinElmer, Waltham, Massachusetts, United States), according to the manufacturer’s instructions. Photon emissions from the obtained luciferase-expressing TC71 (TC71 Luc) cells were measured using the IVIS Lumina XRMS (IVIS Lumina XRMS In Vivo Imaging System, PerkinElmer) with Living Image software (version 4.3.1, PerkinElmer), which was used to estimate the level of photon emission per cell. The MIGR1 vector, which encoded the red fluorescent protein DsRed, was used to stably transduce TC71 Luc cell line to facilitate the distinction of ES cells from MSCs on the VITVO50 matrix. Retrovirus production was performed using the FLYRD18 packaging cell line (RRID:CVCL_8871), as previously described (Marx et al., 1999 (link); Grisendi et al., 2015 (link)).
MSCs were genetically modified with a pCCL PGK WPRE lentiviral vector encoding green fluorescent protein (GFP), as previously reported (Rossignoli et al., 2019 (link)).