when measuring dna content histograms in flow cytometer with DAPI, I often observe a shift of the whole cell cycle distribution to the left or to the right when changing from one sample to another. why is this, how can I avoid this effect? my samples are all treated the same way (same culture method, same fixation and permeabilisation method etc).

Answer

DNA content cell cycle analysis requires careful optimization of every step in the process. Under ideal staining conditions, all cells with the same DNA content are expected to be uniform in staining. However in practice, variation can result from differences in sample prep, staining, methods of acquisition and anaylsis, along with performance of instrumentation. For a given experiment, each sample should contain the same number of cells, as sample to sample variation in cell number leads to significant differences in fluorescence signal. Having a single cell suspension is important is cell staining, as cell clumping or aggregation limits the accessibility of the dye to all cells equally. Methods to remove aggregation before staining include trituration and filtration. Methods used in analysis include as pulse-processing used to remove cell aggregates thru gating and software modeling used to eliminate cell aggregates and debris from the analysis. With hydrodynamic focusing cytometers, acquire in a low flow rate for the most precise results. During the acquisition, you may try waiting a few seconds to allow stabilization of each sample before recording.