Easycyte6ht 2l flow cytometer

Both GFP-HIV-infected and un-infected CEM-T4 cells prepared as described above under “Generation of latent GFP-HIV-infected CEM-T4 cells” were incubated with 200µM of either BAX-inhibitor-V5 or BAX-Inhibiting Peptide Negative Control (Millipore, Billerica, MA) for three hours, then treated with the following drugs (final concentrations): SAHA (350nM); Acitretin (5 µM); equivalent DMSO input. After treatment for 24 hours^{39 (link)}, the cells were stained with Alexa Fluor 647 Annexin V (Life Technologies, Grand Island, NY) to detect apoptosis (100,000 cells per sample) using the EasyCyte6HT-2L flow cytometer (Millipore, Billerica, MA) according to the manufacturer’s instructions.

The following assays were performed in duplicate with the EasyCyte6HT-2L flow cytometer (Millipore, Billerica, MA) according to the manufacturer’s instructions: (1) Guava ViaCount assay (Millipore, Billerica, MA) for absolute total cell counts and percentages of apoptotic cells. (2) Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Life Technologies, Grand Island, NY) to detect apoptosis for all experiments except those using GFP-HIV virus (50,000 cells per sample at each time point) (Fig. 1 and Fig. 3). (3) CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Life Technologies, Grand Island, NY) to detect caspase-3 and caspase-7 activity (50,000 cells per sample) (Fig. 1). (4) Alexa Fluor 647 Annexin V (Life Technologies, Grand Island, NY) to detect apoptosis in GFP-HIV-infected CEM-T4 cells (100,000 cells per sample) (Fig. 2), since GFP would interfere with Alexa Fluor 488 annexin V apoptosis and caspase 3/7-green flow cytometry assays.