Gata1^low BMMCs generate with high efficiency trilineage growth factor-dependent cell lines. (A) SN1, a representative clonal cell line obtained in BMMCs seeded with marrow cells from Gata1^low mice, expresses morphologic markers of the mastocytic (Alcian blue) and megakaryocytic (acetylcholine esterase A) lineage. The procedure to isolate the SN1 cell line is described in “Results.” By May-Grünwald-Giemsa (B1) staining, SN1 cells were large and contained several cytoplasmic granules, that reacted with acetylcholine esterase A (B2) or Alcian blue (B3). Original magnification, × 64. Images were obtained with a Leica DMLS2 microscope equipped with an N PLAN 10×/2.1 objective lens at 10×/0.05 magnification (Leica, Solms, Germany). Photographs were taken with a Leica DFC 280 digital camera, and were acquired with Leica IM50 4.0 software. Subsequent image processing was performed with Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA). (B) The SN1 cells are growth factor dependent and require, for optimal growth, SCF and IL-3. Growth factor requirement was measured by counting the number of cells present after 5 days in cultures supplemented with the growth factors indicated on the abscises. The detailed growth curve of cells in cultures stimulated with SCF and IL-3 is presented in the insert. Results are presented as mean (± SD) cell numbers observed in at least 3 independent experiments. (C) Antigenic profiling of SN1 cells. The different panels present, in the order, dot plot analysis for side-scatter, c-Kit expression and gate used to analyze, in the following histograms, the expression of CD34, SCA-1, and FcϵRI, as indicated. In these histograms, the gray area corresponds to SN1 cells labeled with the antibody indicated on the abscissa, the dotted lines present the profile of serosal Gata1^low mast cells labeled with the same antibody (positive control), and the straight lines correspond to SN1 cells labeled with an irrelevant antibody (negative control). (D) Levels of serotonin (as percent of total serotonin uptake) released on IgE + αIgE stimulation by SN1 cells. Positive and negative controls were represented by cells stimulated with IgE alone, medium + αIgE; medium + HSA + DNP and ionomycin, as indicated. The levels of total serotonin that had been incorporated by the cells was measured by Triton lysis. Although low, the levels of serotonin released on αIgE-IgE stimulation are statistically (P < .05) different from those released on stimulation with IgE alone. Results are presented as the mean (± SD) of at least 3 separate experiments performed in triplicate. (E) Expression profiling of SN1 cells by semiquantitative RT-PCR. The gene analyzed in each reaction is indicated on the right. The triangle on the top of the panels indicates increasing numbers of cycles. The levels of expression are compared to those measured in parallel experiments using, as template, cDNA from a mastocytic (32D)³⁵  and an erythroid (32D EPO) cell line.³⁶  Similar results were observed with 3 additional clonal cell lines obtained from Gata1^low BMMCs (not shown).