Figure 1.

Characterization of the P6C cells. (A) Holoclone formation of the P6C cells. Cells from primary colorectal cancer tissue and paired normal colon tissue were trypsinized and seeded into a 6-well plate. A primary clone from a single cancer cell and a holoclone of the P6C cells (passage 20) are shown in the top column, respectively. A representative primary sphere from the colon cancer tissue and dispersed normal colon cells are shown in the bottom column. Scale bars, 200 μm. (B) Expression of distinct markers in the P6C cells. Suspended P6C cells were incubated with anti-CD45-FITC, CD31-FITC, and CD24-FITC, respectively, and were analysed by flow cytometry. Donkey anti-mouse-FITC was used as a control. M1, negative; M2, positive. (C) Surface expression of CD44 in P6C cells as detected by immunofluorescence. P6C cells were grown attached to plates for 5 d, fixed with paraformaldehyde and incubated with anti-CD44 antibody. DAPI was used to stain the nucleus. Scale bar, 100 μm. (D) Relative expression levels of distinct markers under different culture conditions, including a P6C sphere, a P6C clone and SW480 cells. Suspended cells were collected and incubated with FITC conjugated antibodies and were analysed by flow cytometry. Each sample was analysed in triplicate, and the experiment was repeated 3 times. ^bP<0.05, ^cP<0.01. (E) Comparison of the clonal formation of the P6C, HCT116, and SW480 cell lines. A small number of cells (200) were cultured in 6-well plates for 20 d, and the resulting clones were stained with crystal violet (left). The results of the statistical analysis are shown in the right column. Each cell was seeded in triplicate, and the experiment was repeated 3 times. ^cP<0.01.