American Society of Hematology

Figure 3

$Figure 3. Viability, proliferation, and clonogenicity of sorted SP and MP cells. (A) Viability of sorted SP and MP cells at 1, 3, and 5 days was assessed by MTT. (B) Proliferation of sorted SP and MP cells was assessed by BrdU incorporation at 1 and 3 days. Percentage of cells in undivided generation (origin; undivided cells immediately after sorting measured), “1st” generation (once divided), and “2nd” generation (twice divided) are shown. (C) Viability of sorted CFSE-labeled SP and MP cells both alone or with unlabeled HS-5 BMSCs at 1, 3, and 5 days, was measured using PI staining and analyzed using a FACSCanto II flow cytometer. (D) Proliferation of sorted CFSE-labeled viable SP and MP cells is indirectly proportional to the number of cell divisions, quantified as mean of cell membrane-bound CFSE fluorescent intensity. Fluorescence intensity of CFSE+PI−-labeled SP and MP cells decreases in a time-dependent manner (1, 3, and 5 days). (E) Percentage of SP fraction in 5 MM cell lines (KMS-11, OCIMy5, OPM1, RPMI-Dox40, and RPMI-MR20) at 1, 3, and 5 days. (F) Sorted SP and MP cells cultured for 3 and 5 days were restained with Hoechst 33342. (G) The number of colonies of sorted SP and MP cells was assessed at day 14. Representative images of colonies of sorted SP and MP are shown (using an inverted Leica microscope with a Leica DFC300Fx camera (4×/0.1 and 10×/0.22; Leica IM50 image-acquisition software Version 4).$

Viability, proliferation, and clonogenicity of sorted SP and MP cells. (A) Viability of sorted SP and MP cells at 1, 3, and 5 days was assessed by MTT. (B) Proliferation of sorted SP and MP cells was assessed by BrdU incorporation at 1 and 3 days. Percentage of cells in undivided generation (origin; undivided cells immediately after sorting measured), “1st” generation (once divided), and “2nd” generation (twice divided) are shown. (C) Viability of sorted CFSE-labeled SP and MP cells both alone or with unlabeled HS-5 BMSCs at 1, 3, and 5 days, was measured using PI staining and analyzed using a FACSCanto II flow cytometer. (D) Proliferation of sorted CFSE-labeled viable SP and MP cells is indirectly proportional to the number of cell divisions, quantified as mean of cell membrane-bound CFSE fluorescent intensity. Fluorescence intensity of CFSE⁺PI⁻-labeled SP and MP cells decreases in a time-dependent manner (1, 3, and 5 days). (E) Percentage of SP fraction in 5 MM cell lines (KMS-11, OCIMy5, OPM1, RPMI-Dox40, and RPMI-MR20) at 1, 3, and 5 days. (F) Sorted SP and MP cells cultured for 3 and 5 days were restained with Hoechst 33342. (G) The number of colonies of sorted SP and MP cells was assessed at day 14. Representative images of colonies of sorted SP and MP are shown (using an inverted Leica microscope with a Leica DFC300Fx camera (4×/0.1 and 10×/0.22; Leica IM50 image-acquisition software Version 4).

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