Necrotic cell death as measured by cells staining positive for annexin and PI was enhanced by 8-Cl-cAMP under conditions of IL-3 withdrawal.
Mitochondrial membrane potential was measured after cell staining with 10 µM Rhodamine 123 (Rho123) for 30 min at 37°C.
Background fluorescence was measured using cells stained with secondary antibody only.
To measure the percentage of cells staining positive for Annexin V, the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) was utilized per the manufacturer's instructions.
Next, we determined whether bexarotene induced apoptosis by measuring the proportion of cells staining positive for cleaved caspase 3.
Neither could we find a significant difference in proliferation, measured as fraction of cells staining for cyclin A, between the deceased and the survivors.
Furthermore, apoptotic indices, measured by the percentage of cells staining positively for cleaved caspase 3, were approximately 3 4-fold higher in the two tumors that arose in the p21 −/− mice (per × 400 field: tumor A, 6±2; tumor B, 8±4) compared to wild-type mice (per × 400 field: 1±1).
We used the 'quick score', a simple combination of the proportion of cells staining plus a measure of intensity of staining [ 21].
To quantitatively measure cell mortality, FUN-1 staining was used to distinguish between dead and living cells.
Minocycline did not result in monocyte cell death as measured by LIVE/DEAD cell staining (data not shown).
