Intracellular Staining, Apoptosis, TMRM Assays and Flow Cytometry

Cells (2×10⁵ cells/sample) were treated for 20 min in complete medium at 37°C as above, washed with PBS and fixed in 100 μl of fixation buffer (eBiosciences, #420801) for 15 minutes at RT. Cells were then washed with PBS added with 1% BSA (AppliChem PanReac, #A6588) and incubated with 10 μl permeabilization buffer (eBiosciences, #421008) containing either mouse anti-Bax (B-9) (Santa Cruz Biotechnology Inc., #sc-7480) or rabbit anti-phospho-SHP-1 Tyr564 (Cell Signaling, #D11G5) antibodies at RT for 1 h, washed twice in PBS 1% BSA and then incubated with 10 μl permeabilization buffer containing Alexa Fluor anti-mouse-488 (Thermo Fisher Scientific, #A11001) or anti-rabbit-488 (Thermo Fisher Scientific, #A11008) secondary antibodies for 45 min. After washing with PBS 1% BSA, cell pellets were resuspended in 200 μl PBS 1% BSA and subjected to flow cytometric analysis. Early apoptotic cells were quantified by flow cytometric analysis of 1×10⁶ cells stained with FITC-labeled Annexin V (e-Bioscience, #88-8005-74) and Propidium iodide (PI, 20 µg/mL, Biotium, #40017). Mitochondrial membrane potential was measured using the fluorescent probe tetramethylrhodamine methyl ester (TMRM, Molecular Probes Europe BV). Cells (10⁶ cells/sample) were suspended in 200 μl RPMI-1640 w/o phenol Red (Invitrogen srl) added with 25 mM Hepes pH 7.4 and 200 nM TMRM and incubated for 20 min at 37°C. Cells were then added with 500 ng/ml of the calcium ionophore A23187 (Sigma-Aldrich #C7522), incubated for 10 min at 37°C and subjected to flow cytometric analysis. Flow cytometry was carried out using a Guava Millipore cytometer as described (29). Data were analyzed using Flowjo (Tree Star, Inc.).