Guava personal cell analysis flow cytometer

Flow cytometric analysis was performed using Guava Personal Cell Analysis Flow Cytometer (Merck, Millipore, Burlington, MA, United States) and CytoSoft 2.1.4 software. The assay was performed by seeding 1 × 105 to 1.3 × 105 cells/well. After 24 h of plating, the HAMLET complex was added, and cells were incubated for 6 h in the absence of different HAMLET complex concentrations. After 6 h, cells were detached using trypsin-EDTA without discarding the floating cells. The culture medium was removed by centrifugation, and cells were suspended in a binding buffer. The cells were stained with annexin V-PE and 7-AAD dyes and measured by flow cytometry.

We used the Flow Cellect Mito Damage Kit and Annexin V-PE Apoptosis Detection Kit obtained from EMD Millipore in the United States. To perform the flow cytometric analysis and understand how cells responded to HAMLET, we seeded 100,000 to 130,000 cells per well. After one day, we treated these cells with different concentrations of HAMLET for 6 h. We then detached the cells, stained them with Annexin V-PE and 7-AAD, and analyzed them using the Guava Personal Cell Analysis Flow Cytometer and CytoSoft software (version 2.1.4; Guava; EMD Millipore, Burlington, MA, USA).