Lsrfortessa 14 color

MDA-MB-468 cells stably expressing the mCherry-GFP-LC3 fusion protein were plated in 6-well plates at a density of 6 × 10⁵ cells/well in growth media DMEM/F12 + GlutaMAX-1 (Gibco) supplemented with 10% FBS (Gibco) and 1× Anti-Anti (Gibco) and allowed to grow overnight. Cells were treated, trypsinized, and then resuspended in PBS, and analytical cytometry was performed in the Sanford Burnham Prebys flow cytometry core using an LSRFortessa 14-color (BD Biosciences) with a 488 nm and 610 nM laser. For flow cytometry, WT MDA-MB-468 cells were used as the unstained control, and MDA-MB-468 cells expressing either mCherry or GFP were used for compensation. Events of 10,000 were collected for each sample.

MDA-MB-468 cells were treated with compound 26 or DMSO for 18 h after which cells the were trypsinized and washed twice with cold PBS. The cells were then stained using a PE Annexin V Apoptosis Detection Kit I (BD Pharmingen #559763). Specifically, the cells were resuspended in 1 × binding buffer at a concentration of 1 × 10⁶ cells/mL, and 100 μL of the solution was transferred to a5 mL culture tube. PE Annexin V (5 μL) and 7-AAD (5 μL) were added to the tube with cells. The mix was incubated for 15 min at room temperature in the dark after gentle vortexing. Next, 1× binding buffer (400 μL) was added to each tube, and flow cytometry to detect PE-Annexin V and 7-AAD was performed within 1 h using a LSRFortessa 14-color (BD Biosciences) to analyze apoptosis. Events of 10,000 were collected for each sample.