We are developing an assay to measure an analogue for cell transfection. In theory, DAPI (blue) allows us to locate all cells, Calcein AM (green) indicates viability and Propidium Iodide (red) indicates a compromised membrane. Thus, a blue, green and red fluorescent object would be a viable and membrane-permeabilsed cell. Is there anything wrong with this logic? Would these dyes quench each other?
Dear Ryan
Dapi is membrane permeable and stains all nuclei, but Dapi also stains cells with a permeabilised plasma membrane quicker than cells with an intact plasma membrane.
Calcein-AM will be trapped inside living cells when the AM group is cleaved off in the cytosol which renders the Calcein membrane impermeable. Permeabilisation of the membrane will lead to a loss of at least some of the calcein. Also calcein is usually exported by multi drug resistance transporters, so it might not stay for more than a few hrs, depending on your cells.
PI is membrane impermeable and binds to DNA when the plasmamembrane is permeabilised, staining might take 30min. Dapi might be slightly quenched when high amounds of PI bind to the DNA.
Usually: DAPI + PI = dead, DAPI+Calcein = alive
I would call a cell with a permeabilised plasma membrane not alive unless it was only a transient permeabilisation e.g. due to electroporation.
Best
Heiko
Dear Heiko,
Thank you for your information. By regards to your information, so if I stain the non-fixed cells with PI in a cell plate as a marker of dead cells and then fixed cells and stain them with DAPI, will I have dead cells with red color and all cells with blue cells?
Hi Ryan, hi Shima
I have to apologize, not to recommend Hoechst over DAPI for live cells. DAPI will only stain live cells if used at high concentrations.
If you swap DAPI with Hoechst (e.g. 1ug/ml) the answer above is correct, otherwise do like Shima recommends.
There will be some quenching of Hoechst/DAPI by PI in dead cells but both dyes will stay visible.
Note: There are differences Hoechst nuclear stain spectral properties: 33342/33258 and 34580. The first 2 are better to multiplex with calcein/GFP/FITC etc (even CFP works) the latter is better for excitation with the 405 nm laser.
sorry again
Heiko