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The key elements in obtaining a histogram of high quality are good sample preparation, correct cell-to dye ratio and proper instrument alignment. Live cell histograms are more challenging to generate than fixed cell ones.
- Make sure the cell preparation protocol generates single cells with minimal clumping. Filtration or trituration of cell suspensions may be required.
- Systematically vary the dye-to-cell ratio.
- Optimize for cell type, medium or buffer used, time of incubation, temperature of incubation.
- Gate on the live cells and increase the number of cycle events being analyzed.
- Verify that the flow cytometer is correctly aligned, with stable fluidics, using calibrated fluorescent beads of known CV.
- Verify that the cell concentration is 1 x 105 to 5 x105 (red dye) or 1 x 106, (green dye) cells/mL.
- For permeabilized cells ensure that sufficient Triton X-100 has been added to the staining solution.
- For ethanol fixed cells ensure that the cells were fixed and stored correctly.