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. 2021 Jun 24;22(13):6802. doi: 10.3390/ijms22136802

Figure 4.

Figure 4

Multicolor flow cytometry analysis of fetal hemoglobin expression by K562 cells. At the end of incubation, K562 cells were harvested for analysis to determine the median FSC-A by flow cytometry. To exclude dead cells from the analysis, the cells were first stained with FVS450. After fixation (BD Cytofix/Cytoperm Buffer) and permeabilization (BD Perm/Wash Buffer), the cells were stained with PE Mouse Anti-Human Fetal Hemoglobin (FHb) antibodies (clone 2D12) or the corresponding isotype. (A) Presentation of gating strategy for excluding debris and dead cells from the analysis of the intracellular expression of FHb. Forward and side scatter (FSC, SSC) plot show debris at the left bottom corner of the FSC vs. the SSC density plot. The dot plot in the middle panel shows the incorporated levels of FVS450 versus SSC. Events with a low level of FVS450 incorporation represent live cells. Sample histograms (right panel) show the levels of FHb expressed by live cells. uK562—undifferentiated K562 cells, unstained (negative control), isoK562—undifferentiated K562 cells, stained with isotype (isotype control), uK562 FHb—undifferentiated K562 cells, stained for FHb, dK562 FHb—differentiated K562 cells stained for FHb. (B) Sample FSC-A histograms show the decrease in cell size of undifferentiated (left panel) and differentiated (right panel) K562 cells after everolimus stimulation. Flow cytometry was performed using a BD FACSLyric™ Flow Cytometer System.