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. 2014 Apr 9;4:4619. doi: 10.1038/srep04619

Figure 5. Flow chart of the study showing the experimental design.

Figure 5

Primary cells from nasal tissues were isolated and seeded on 3T3 feeders on Day 0 in passage 0 (P0). The culture conditions favor human nasal epithelial stem/progenitor cells (hNESPCs), therefore, other cell types like fibroblasts, lymphocytes, and epithelial differentiated cells could not grow in this system. After 48 hours, 10 random colonies were marked and tracked for 3 days. Observation of the cell morphology and evaluation of doubling time were performed on T1, T2, and T3. On T3, the cells in three wells of the 24-well plate were fixed and stained for calculation of CFE and immunofluorescence assay. On the day of cell confluence (Day 6), the hNESPCs in P0 were enzymatically digested and subcultured. Cellular RNA was also obtained at this point. Hereafter, the cells were continually passaged to P3. The same observation and evaluation procedures were also done in P1, P2, and P3.