I am working with blood and A549 cancer cell. My experiment design is where I will stain the cancer cells with CellTracker CMTPX, and also stain the blood (lysed blood sample with a few residual RBC) with hoechst separately and then spike the stained blood sample with the stained cancer cells.
There are no issues with the dye used for cancer cell but the hoechst always stains the cancer cells a dull blue as well after I spike the blood with cancer cells. I have tried to wash the stained blood cells 5 times in PBS to prevent this stain contamination but it keeps happening. Is there any way to prevent this?
I think the stained blood sample has residual Hoescht and then since Hoechst is cell-permeable the cancer cells stain with residual Hoechst. A few follow-up questions to help.
What is the concentration you are using?
What is the purpose of staining with Hoechst? If it is just to stain the DNA (does not matter if it is dead or alive), then I do not see an issue with the blue color.
Hoechst is cell-permeable. If you are using it on live cells, you would want to use something else that is not permeable without fixation or if the cell is dead, such as Propodium Iodine (PI). What color is your CellTracker? Make sure that the PI and CellTracker excitations do not overlap.
Hoechst stains the nucleus (DNA there), so it needs intact cells with a nucleus. If a blood sample is lysed, as you are saying, then cells are fragmented with nuclei. I think the lack of intact cells in blood is the reason for the issue you are getting with blood staining, as there is no issue with A549 cells. I hope I could have understood your question.
Dear Nusaibah Reza ,
The Hoechst dye (I assume you are using 33342) is not bound permanently in your cells. It's a DNA-intercalating Dye. So that you have always also unbound free Hoechst in the cell (for example because of conformation changes of the DNA during transcription) and since it's cell(membrane)-permeable it must stain co cultured cells.
You should switch to a dye which is bound (via reactive groups like AM) permanently in the cells.
Best wishes Soenke
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