What method/assay was used to make figure 2? In figure 2, what do the two cell c

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What method/assay was used to make figure 2? In figure 2, what do the two cell clusters represent? What is propidium iodide and Annexin V?

Figure 2. Metformin induces CD19-CAR T-cell apoptosis. (A) CD19-CAR T cells were treated with metformin (0, 1, 10, and 20 mM) for 24 hours. The apoptotic rate of CD19-CAR T cells was detected by flow cytometry. (B) Statistical analysis of the flow cytometry results. The results are represented as mean ± SD from three independent experiments. *P,0.05, **P,0.01 versus the control group.

Metformin induces CD19-CAR T-cell apoptosis To determine whether metformin has a direct effect on CD19-CAR T-cell apoptosis, the cells were incubated with metformin (1, 10, and 20 mM) for 24 hours. As shown in flow cytometry results, metformin (1, 10, and 20 mM) effectively induced CD19-CAR T-cell apoptosis (Figure 2A and B). These data demonstrated the pro-apoptotic role of metformin on CD19-CAR T cells.

Control Met (1 mM) 104 104 103 103 11.0% 10% 10% 40 101 101 6.11% 7.36% 100 10° 101 102 103 104 100 101 102 103 104 100 ? 20 0 Met (10 mM) Met (20 mM) 104 104 ? Q 10 103 103 0 13.1% 20.3% 102 102 101?! 101 10.1% 6.96% 100 100 100 101 10% 10% 10% 100 101 102 103 104 Annexin V

Explanation / Answer

In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a phospholipid binding binding protein and binds to the PS exposed on the outer surface of the apoptotic cells. Cell membrane are impermeable to propidium iodide (PI). So in oreder to analyse and seperate live and dead cell PI staining can be done. A live intact cell will not show a PI staining however the cell which are in the late apoptotic phase or are dead will be have a PI staining.

Based on the this priciple the above figure shows the Annexin V and PI staining. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Therefore, cells that are considered viable are both Annexin V and PI negative, while cells that are in early apoptosis are Annexin V positive and PI negative, and cells that are in late apoptosis or already dead are both Annexin V and PI positive.

Here the lower left quadrant shows PI and annexin V negative cells(live intact cells). The lower fight quadrant shows PI negative and annexin positive stained cells (early apoptosis). The upper left quadrant shows PI positive and annexin V negative cells (damaged cell membrane/ dead cells). The upper right quadrant shows PI and annexin V positive cells which are in the late apoptotic or dead stage. Through this process it is not very clear to differentiate between late apoptotic and dad cells Since both are PI and Annexin V positive.

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