Mitotracker cmxros red fluorescent dye

Single-cell suspensions were prepared and stained using standard techniques. Cells were fixed with eBio Fix/Perm kit to stain for intracellular transcription factors. To detect intracellular phospho-Akt, cells were treated sequentially with 3% paraformaldehyde, ice-cold methanol, and eBioscience 1x permeabilization buffer prior to antibody staining. Mitochondrial membrane potential was measured by incubating cells with MitoTracker CMXRos red-fluorescent dye (Cell Signaling) for 30 min. FACS antibodies are listed in Supplemental Experimental Procedures.
BD LSRII/Fortessa and FACSAria II flow cytometers with FACSDiva software were used to analyze and/or purify cells. Data were analyzed using Flowjo v.8.8.7 (Treestar). Each FACS plot axis displays increasing fluorescence intensity of the labeled molecule. The cell division x-axis label has an inverse (leftward) arrow, denoting that intensity of fluorescent dye covalently bound to intracellular proteins undergoes dilution with each successive cell division. Within FACS plots, each dot represents a single cell and numbers displayed adjacent to bound-areas (gates) represent the frequency of cells within the gate.

Purified naive B cells labeled with CFSE, CTV, or eFluor670 cell proliferation dyes were cultured in lipopolysaccharide (LPS-SM ultrapure; Invivogen; 10–25 μg/mL) in 48 well plates at 2.5 × 10⁵ cells/mL of culture media in a 37 °C humidified environment with 5% CO₂. To activate CD8⁺ T cell in vitro, wild-type or P14⁺ CD8⁺ T cells were purified, labeled with cell proliferation dye and stimulated with immobilized anti-CD3 (5 μg/ml), soluble anti-CD28 (1 μg/ml) and recombinant IL-2 (30 IU), or by gp33 (1 μg/ml; Anaspec) peptide-loaded splenocytes, respectively. Memory CD8⁺ T cells were harvested at 120⁺ days after LCMV infection.
Small molecule inhibitors of PI3K (LY294002; Cell Signaling), pan-AKT (Triciribine; Cayman Chemical Company), mTOR (Rapamycin; Selleckchem), and FoxO1 (AS1842856; Calbiochem) were added to the cells at the time of activation. Mitochondria membrane potential was measured by incubating cells with MitoTracker CMXRos red-fluorescent dye (Cell Signaling) for 30 minutes followed by washing cells three times with ice-cold culture media.