Mast cell progenitors mature in the absence of SCF. (A) The CD34-enriched progenitors were purified from buffy coats and cultured with IL-3 and IL-6. The FSC and SSC parameters of the primary mast cell progenitors (CD117^int/hiFcεRI⁺ cells, left) and the cultured pre–mast cells (CD117^hiFcεRI⁺ cells, right) are shown in black height curves. The gray height curves indicate unstimulated CD34⁺ cells. One representative buffy coat out of 3 is shown. (B) The mean fluorescence intensities of the FSC and SSC parameters were calculated for each population described in panel A. The ratio between the CD117^int/hiFcεRI⁺ cells and the unstimulated CD34⁺ cells was then calculated for each time point independently, as the photomultiplier tube voltages of those parameters were adjusted between day 0 and 5 to visualize the cells on scale. The results were pooled from 3 buffy coats. The bars represent the means ± SEM. Unpaired 2-tailed Student t tests. **P < .01; ***P < .001. (C) CD117⁺ cells were enriched from buffy coats, and CD34⁺CD117^int/hiFcεRI⁺ cells were isolated with FACS. The isolated CD34⁺CD117^int/hiFcεRI⁺ cells were stained with May-Grünwald Giemsa. (D) Sorted CD34⁺CD117^int/hiFcεRI⁺ cells were cultured with IL-3 and IL-6 for 5 days. The cultured cells were analyzed with May-Grünwald Giemsa staining, an enzyme cytochemical staining for trypsinlike activity as a measure of tryptase, and flow cytometry. Live singlet cells are shown in the flow cytometric graph. (E) Sorted CD34⁺CD117^int/hiFcεRI⁺ cells were first cultured with IL-3 and IL-6 for 5 days and then cultured with SCF and IL-6 for an additional 5 to 7 days. The cultured cells were analyzed with May-Grünwald Giemsa staining and an enzyme cytochemical staining for trypsinlike activity as a measure of tryptase. (F) The largest cell diameter of the primary mast cell progenitors, pre–mast cells, and mast cells was measured using ImageJ (NIH, Bethesda, MD). Each dot in the graph corresponds to 1 cell. The means ± SEM are shown. One-way ANOVA with Tukey’s multiple comparisons test. ***Adjusted P < .001; ****adjusted P < .0001. Images were captured using the Eclipse E400 microscope, the Digital Camera DXM1200, and ACT-1 software (Nikon, Tokyo, Japan). The width of the May-Grünwald Giemsa photos corresponds to 28 μm. The width of the enzyme cytochemical staining photos corresponds to 35 μm. The cultures at day 5 and days 10 to 12 were performed for 2 buffy coats with similar results. The day 0 May-Grünwald Giemsa staining was performed for 1 buffy coat.