SCID mice were routinely euthanized at day 5 after surgery and border zones of hearts were removed and digested into single cells. The single cells were stained by H2-Kd and dyecycle (Invitrogen), and then subjected to FACS. MSCs isolated from donor transgenic mice were H2-Kd negative, whereas recipient SCID mice were H2-Kd positive. In addition, dyecycle staining was capable of distinguishing sing cell or fused cell via DNA content, and 2N was considered as a marker of single cell.
Inventoried TaqMan Assays (20×, Applied Biosystems) were pooled and diluted to a final concentration of 0.2× for each of the 24 probes. Individual cells were directly collected into 10 μl RT-PreAmp MasterMix (5.0 μl CellsDirect 2× Reaction Mix [Invitrogen]; 2.5 μl 0.2× assay pool; 0.5 μl RT/Taq enzyme [CellsDirect qRT-PCR kit, Invitrogen]; 2.0 μl TE buffer). The products were analyzed with Universal PCR Master Mix and inventoried TaqMan gene expression assays in 48.48 Dynamic Arrays on a BioMark System (Fluidigm, CA, USA). CT values were calculated from the system’s software (Fluidigm). The resulting values were then normalized to the endogenous controls by subtracting the average of GAPDH expression levels.
Inventoried TaqMan Assays (20×, Applied Biosystems) were pooled and diluted to a final concentration of 0.2× for each of the 24 probes. Individual cells were directly collected into 10 μl RT-PreAmp MasterMix (5.0 μl CellsDirect 2× Reaction Mix [Invitrogen]; 2.5 μl 0.2× assay pool; 0.5 μl RT/Taq enzyme [CellsDirect qRT-PCR kit, Invitrogen]; 2.0 μl TE buffer). The products were analyzed with Universal PCR Master Mix and inventoried TaqMan gene expression assays in 48.48 Dynamic Arrays on a BioMark System (Fluidigm, CA, USA). CT values were calculated from the system’s software (Fluidigm). The resulting values were then normalized to the endogenous controls by subtracting the average of GAPDH expression levels.