I am conducting an experiment where I am putting cultured cancer cells (B-ALL line) in whole blood and running them through a microfluidic device. I stain the cells with DAPI and DiI (cell membrane dye) and then add them to whole blood. The issue that I am facing is that clumps develop in my microfluidic device when I run the sample. I have noticed that the amount of clumps and the time it takes for them to develop are related to the concentration of cancer cells I add in the blood. (The more cells I add, the bigger the clumps and the quicker they develop.) Does anyone have experience with spiking cancer cells in whole blood and recommend any changes? Should I fix the cancer cells before spiking?
Dear Mubasher Iqbal ,
Greetings.
So, if i suggest you to do few things.
First; Your total concentration of blood+cells should not exceed than 1M cells/ml for appropriate distribution.
Secondly; Clumping is not just because of cancer cells (or their concentration) but PI cause aggregation. If you may use any other DNA dye, it will save you further. PI is known to be very sticky dye. even in Flowcytometry, people need to clean the probe b/t the sample to remove PI carryover.
Then, cancer cells tends to form aggregates (called blasts). So you need to add a bit more anticoagulant in the buffer.
Finally, if nothing works; use 20-30 uM nylon mesh to clear aggregates before running the sample in instrument.
I hope your query is answered.
Best wishes.
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