KLF1-GATA1 fusion protein reduces hypoxia-related sickling in erythroid cells cultured from SCD mouse HSCs. Sca-1⁺c-Kit⁺Lin^/low HSCs isolated from SCD mice were transduced with vector only, KLF1, or KLF1-GATA1 fusion protein, then grown in a 3-phase erythroid culture system. (A–D) SIFCA of cultured erythroid cells stained with TER119-APC antibody and TO. (A) Bright-field and fluorescence images of erythroid cells acquired by imaging flow cytometry, illustrating examples of the cell morphologies. (B) SIFCA identifying the shape ratio cutoff of 0.5 used for classifying cultured erythroid cells as normal/round, abnormal/sickled, or undefined/indeterminate, and associated representative bright-field imagery. Shape ratio is the minimum width of the cell divided by the maximum length identified by the analysis software. Cells with a shape ratio lower than the cutoff values were designated as “abnormal cells (sickled cells).” Cells with a shape ratio higher than the cutoff level were defined as “normal cells (non-sickled cells).” Cells were designated as “undefined” if the cells could not be categorized.^87,88 (C) Representative SIFCA of cultured erythroid cells. (D) Graphic representation showing the mean percentage of normal cells identified through SIFCA. ∗∗P < .01 vs vector control–transduced cells. Error bars indicate the mean ± SD of 3 independent experiments. (E) Representative flow-cytometric analysis of cultured erythroid cells stained with TER119-APC antibody and TO to assess erythroid enucleation. Data represent the mean ± SD of 3 independent experiments. (F) Giemsa staining of cultured erythroid cells. Original magnification, ×40. All analyses were performed at the time points indicated in Figure 4B. KG-M, KLF1-GATA1 medium-form; KG-L, KLF1-GATA1 long-form; SIFCA, sickle cell imaging flow cytometry assay.