The kit contains a mouse monoclonal anti-phospho-Rb primary antibody specific for Rb phosphorylated at Ser608, a goat anti-mouse secondary antibody conjugated to the DyLight 549 Fluorophore, DAPI dye to identify the cell cycle phase by DNA content and various other reagents and buffers required for immunofluorescence labeling of cell cycle specific Rb phosphorylation for high-content screening (HCS) assays.
Rb, the retinoblastoma susceptibility gene, was identified as the first tumor suppressor gene. The activation of Rb is controlled via multiple phosphorylation by cyclin/Cdk complexes (especially the cyclin D and Cdk4/6 complex) during the cell cycle. When hypophosphorylated, Rb inhibits cell cycle progression by repression of E2F transcription factor until the restriction (R) point in G1 phase. (The R-point is the time position in late G1 phase after which a cell is committed to undergo DNA replication and is no longer sensitive to growth factors until the cell division.) After the R-point in G1, Rb becomes hyperphosphorylated, thereby activating E2F transcription factor and facilitating cell cycle progression. Rb is again dephosphorylated at the end of mitosis. The activity of phospho-Rb is closely associated with its nuclear intensity of phospho-Rb. The activation of phospho-Rb can be monitored by the nuclear intensity change of phospho-Rb during the cell cycle. Rb is also known to perform specific roles in differentiation, senescence and apoptosis.
Figure 2. Phospho-Rb staining in A549 lung carcinoma cells. A549 cells were either non-treated or treated with Nocodazole (0.5 μg/ml, 16 hours), then fixed and stained according to the kit procedure with anti-phospho-Rb primary antibody, DyLight 549 Goat Anti-Mouse Secondary Antibody and DAPI dye. The left panels are nuclear staining with DAPI; the right panels are phospho-Rb staining in the same cells. The intracellular level of phospho-Rb increases when cell cycle is blocked at G2/M phase with Nocodazole treatment (i.e., have 4N DNA content and ready to undergo mitosis). One mitotic cell in the non-treated panel also shows increased intensity in phospho-Rb staining.
Figure 3. Color-merged images of DAPI (blue) and Phospho-Rb (orange; rendered green) channels in non-treated (control)(left) and treated cells (right). Dose response curves of phospho-Rb for three drug compounds that affect the cell cycle (bottom). Drugs were added to cells at the indicated concentrations. Each data point represents eight wells from one to three plates. Phospho-Rb was measured using the output parameter of the nuclear average fluorescence intensity (MEAN_CircAvgIntenCh2) with Cellomics Compartmental Analysis BioApplication. Values are normalized with the maximum control value and presented as % Control.
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