Life Technologies

Calculation of reprogramming efficiency At day 27 of reprogramming, cells were live-stained with Alexa Fluor® 647 dye–conjugated anti-SSEA4 antibody. This antibody detects a common pluripotent stem cell marker and was used to determine reprogramming efficiencies (Table 1). Reprogramming efficiency was calculated as the number of SSEA4-positive colonies divided by the number of cells plated on the same 10 cm dish, 7 days after transduction with the CytoTune®-iPS Sendai Reprogramming Kit. We observed that (1) the reprogramming efficiency varied from line to line, ranging from slightly less than 0.01% to nearly 0.3%, (2) younger patient cells (PD-1) generated more colonies on lower cell plating density dishes than older patient cells, and (3) the observed reprogramming efficiency was not linear with respect to the number of transduced fibroblasts plated per dish. Because of the variability between patient-derived lines observed here, as well as our observations in other studies, we highly recommend plating cells onto MEF dishes at multiple densities on day 7 to ensure the formation of enough single colonies for picking. Colonies from each cell line were expanded for further characterization. Table 1. Reprogramming efficiencies. Day 7 plating density, number of cells plated per 10 cm MEF dish 50,000 cells Cell line Age Gender SSEA4+ colonies 100,000 cells Reprogram efficiency SSEA4+ colonies 200,000 cells Reprogram efficiency SSEA4+ colonies Reprogram efficiency 300,000 cells SSEA4+ colonies Reprogram efficiency PD-1 19 F 55 0.110% 132 0.132% 583 0.292% 658 0.219% Ctrl-1 25 F 35 0.070% 81 0.081% 97 0.049% 161 0.054% Ctrl-2 69 F 22 0.044% 18 0.018% 63 0.032% 75 0.025% PD-2 74 M 55 0.110% 63 0.063% 193 0.097% 357 0.119% MSA 62 F 3 0.006% 20 0.020% 40 0.020% 80 0.027% PD-3 61 M 3 0.006% 7 0.007% 17 0.009% 24 0.008% 13 Life Technologies | Parkinson's cell model

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