Hello! I would like to discuss one problem. For my experiment it was needed to stain alive cells with lipophilic fluorescent dye - PKH2, (it produce green fluorescence), as next step - to incubate with unstained (also alive) cells, next step - to collect in different time intervals, and to check DNA cell cycle distribution with P. Iodide only in stained with PKH2 cells. At first, I wanted to fix cells with 2% of paraformaldehide, then separate cells using sorter on two fraction (PKH2 positive and negative and then to stain with PI and to make DNA analysis by standard protocol, but
1. After fixation with paraformaldehide (PF) DNA profile looks very different to compare with cells that were not fixed with PF, why it is I cannot understand.
2. Like possible solution I can not fix cells with PF, but in such situation I have to run to sorter every six hours several times a day, and may be not only one day.
May be somebody can to recommend me something?
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