I culture multilayered cells, and I found previously that the live nuclear staining dye Hoechst 33342 enters the cells not in contact with the culture medium (cells in the lower layer) very poorly. I am afraid that BrdU would also fail to enter the cells, and I would get a false readout for non-proliferating cells. What other markers (immunofluorescence based) can I use to detect cells that are likely to divide (S-phase)?
Have you tried performing your live labeling with cells grown on transparent filters? Medium with the Hoechst dye can be added above and below the culture for uniform uptake. https://www.corning.com/catalog/cls/documents/protocols/Guidelines_for_Use_Falcon_Cell_Culture_Inserts_PD104401.pdf
Ellen A G Chernoff i want to study the effect of substrate mechanics and need to culture them on glass as well, that's why unable to grow them on a membrane. I am looking at their migration as a collective sheet, so I would not be able to disrupt any cell-cell adhesion either. Are there any specific markers I could use (antibody staining) that will surely tell me which cells are in the S/G2 phase of cell cycle? I used YAP1 as an indicator earlier but that will not tell me the state of cell cycle.
Use antibody for cyclin A. Its expression should be increased in the S and G2 phases of the cell cycle.
For fixed cell S-phase identification I use PCNA antibodies.https://www.sciencedirect.com/science/article/abs/pii/0145212686900214
Article Discrimination of cell cycle phases in PCNA-immunolabeled cells
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