Living image 4

An investigator blinded to the treatment group (RR) analyzed the BLI data using the Living Image 4.4 (Caliper Life Sciences, Massachusetts, USA). Size-constant regions of interest (ROIs) were placed on the ipsi- (right; infarct lesion) and contralesional (left) hemisphere of the head measuring total flux in p/s. To eliminate intra- and interindividual variations of total flux, the ratio between the right and left hemisphere was calculated for each animal individually. To analyze the migration of the hotspot of DCX-labeled cells, BLI images of each time point and animal with the maximum of total flux were identified using Living Image 4.4 (Caliper Life Sciences, Massachusetts, USA). For each animal and each time point, an individualized scale was set to visualize the hotspot. Using the software ImageJ (National Institutes of Health, Bethesda, USA), the respective hotspot was encircled, and the center of the circle was marked. To determine any migration of the hotspot, the distance from the middle of the circle to the midline was measured in mm.

The cell viability of BM-MSCs was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay as described previously [17 (link)]. Briefly, BM-MSCs were plated in 96-well plates at 1 × 10⁵ cells/well. After different treatments, BM-MSCs were incubated with MTT solution (5 g/L, Sigma) at 37 °C for 4 h. The medium was then removed and 200 μL dimethyl sulphoxide (DMSO) was added to each well. The absorbance was determined at a wavelength of 490 nm. Optical density (OD) values for each group were detected in six duplicate wells and their averages were calculated. Furthermore, we also assessed cell viability with bioluminescence imaging (BLI) using the IVIS Kinetic system (Caliper, Hopkinton, MA, USA) [7 (link)]. Briefly, MSCs were plated in 24-well plates (5 × 10⁴ per well). After different treatments, cell culture media were removed. Cells were incubated with D-Luciferin reporter probe (4.5 μg/mL) and then measured using the IVIS Xenogen Kinetic system (Caliper Life Sciences, USA), using the following imaging parameters: binning, 4; F/stop 1; time, 1 min. Bioluminescent signals were analyzed using Living Image 4.0 software (Caliper, MA, USA) and quantified as photons per second per centimeter square per steridian (photons/s/cm²/sr).

Mice were anesthetized by i.p. injection of 80 mg/kg body weight ketamine hydrochloride (Pfizer) and 16 mg/kg xylazine (CP Pharma). Together with anesthetics, mice were injected with 150 mg/kg D-luciferin (Biosynth). After 10 min, BLI signals of the anesthetized mice were recorded using an IVIS Spectrum Imaging system (Caliper Life Sciences). For ex vivo imaging of internal organs 6 d after allo-HCT, mice were injected with D-luciferin and sacrificed 10 min later. Internal organs were removed and subjected to BLI. All pictures were taken with a maximum of 5-min exposure time and analyzed with the Living Image 4.0 software (Caliper Life Sciences) (14 (link), 19 (link)).

BLI was performed to track transplanted BM-MSCs using an IVIS® Kinetic system (Caliper, Hopkinton, MA, USA) [1 (link)]. After intraperitoneal injection with d-luciferin (375 mg/kg body weight), recipient mice were anesthetized with isoflurane and imaged for 10 min on days 1, 7, 14, 21, and 28 until sacrificed. Peak signals (photons/s/cm²/sr) from a fixed region of interest (ROI) were analyzed with Living Image® 4.0 software (Caliper, MA, USA). All these assays were performed in a blinded manner.

Ex vivo lung bioluminescence imaging was performed as previously described (Chopra et al., 2013 (link), 2015 (link)). Briefly, mice were injected with 300 mg/kg D-luciferin and euthanized after 10 min to prepare lungs and immediately subjected to ex vivo bioluminescence imaging with an IVIS Spectrum imaging system (Perkin–Elmer/Caliper Life Sciences, Mainz, Germany). Images were evaluated with Living Image 4.0 software (Caliper Life Sciences).