American Society of Hematology

Figure 2.

$Figure 2. SCF is dispensable for mast cell progenitor survival in vitro. CD34+ progenitors were purified from buffy coats from healthy donors and analyzed with flow cytometry. (A-B) The representative gating strategy and the quantification of CD34+CD117int/hiFcεRI+ mast cell progenitors are shown (n = 9). The representative example in panel A is indicated with an open circle. The line in panel B represents the geometric mean. (C-G) CD34-enriched cells were cultured and analyzed by flow cytometry. (D) The fraction of CD117hiFcεRI+ pre–mast cells from panel C was normalized to the combined IL-3 and IL-6 condition for each buffy coat, and the results from 3 buffy coats were pooled. The bars represent the means ± standard error of the mean (SEM). (E) CD34+ progenitors were cultured with IL-3 and IL-6, and the fractions of CD34-expressing cells out of the CD117hiFcεRI+ mast cell population (days 3-7) are shown. The CD34 expression of CD117int/hiFcεRI+ cells is shown as the day 0 control. The bars represent the means ± SEM of 3 buffy coats. (F) The CD117 expression of CD34+ cells was analyzed on day 3. The red histogram indicates the cells cultured without cytokines. The blue line indicates the cells cultured with IL-3 and IL-6. The dashed line indicates the cells cultured with IL-3, IL-6, and SCF. One representative experiment out of 3 is shown. (G) The CD117 expression on CD34+ cells at day 3, shown in panel F, was quantified by calculation of the median fluorescence intensity. The expression level of CD117 is shown as a percentage of the IL-3 and IL-6 condition. The bars represent the means ± SEM of 3 buffy coats. (H-J) CD34+ cells were enriched from buffy coats, cultured, and analyzed by flow cytometry. The medium was supplemented with polyclonal goat IgG or anti-SCF neutralizing antibodies as indicated. (J) The integrin β7 expression was analyzed by flow cytometry before and after culture with IL-3, IL-6, and anti-SCF. The bars in panels I and J represent the means ± SEM of 3 buffy coats. Live singlet cells are shown in the flow cytometric graphs. The statistical analyses in panels D, E, G, and I were performed using 1-way ANOVA with Tukey’s multiple comparisons test. **Adjusted P < .01; ****adjusted P < .0001; ns = not significant. The unpaired 2-tailed Student t test was used for the statistical analysis in panel J. ****P < .0001.$

SCF is dispensable for mast cell progenitor survival in vitro. CD34⁺ progenitors were purified from buffy coats from healthy donors and analyzed with flow cytometry. (A-B) The representative gating strategy and the quantification of CD34⁺CD117^int/hiFcεRI⁺ mast cell progenitors are shown (n = 9). The representative example in panel A is indicated with an open circle. The line in panel B represents the geometric mean. (C-G) CD34-enriched cells were cultured and analyzed by flow cytometry. (D) The fraction of CD117^hiFcεRI⁺ pre–mast cells from panel C was normalized to the combined IL-3 and IL-6 condition for each buffy coat, and the results from 3 buffy coats were pooled. The bars represent the means ± standard error of the mean (SEM). (E) CD34⁺ progenitors were cultured with IL-3 and IL-6, and the fractions of CD34-expressing cells out of the CD117^hiFcεRI⁺ mast cell population (days 3-7) are shown. The CD34 expression of CD117^int/hiFcεRI⁺ cells is shown as the day 0 control. The bars represent the means ± SEM of 3 buffy coats. (F) The CD117 expression of CD34⁺ cells was analyzed on day 3. The red histogram indicates the cells cultured without cytokines. The blue line indicates the cells cultured with IL-3 and IL-6. The dashed line indicates the cells cultured with IL-3, IL-6, and SCF. One representative experiment out of 3 is shown. (G) The CD117 expression on CD34⁺ cells at day 3, shown in panel F, was quantified by calculation of the median fluorescence intensity. The expression level of CD117 is shown as a percentage of the IL-3 and IL-6 condition. The bars represent the means ± SEM of 3 buffy coats. (H-J) CD34⁺ cells were enriched from buffy coats, cultured, and analyzed by flow cytometry. The medium was supplemented with polyclonal goat IgG or anti-SCF neutralizing antibodies as indicated. (J) The integrin β7 expression was analyzed by flow cytometry before and after culture with IL-3, IL-6, and anti-SCF. The bars in panels I and J represent the means ± SEM of 3 buffy coats. Live singlet cells are shown in the flow cytometric graphs. The statistical analyses in panels D, E, G, and I were performed using 1-way ANOVA with Tukey’s multiple comparisons test. **Adjusted P < .01; ****adjusted P < .0001; ns = not significant. The unpaired 2-tailed Student t test was used for the statistical analysis in panel J. ****P < .0001.

This Feature Is Available To Subscribers Only