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- Incorrect drug concentration or incubation time for cell type:
- Make sure the cell preparation protocol generates single cells with minimal clumping. Filtration or triturating 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.
- 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 1 x 106 cells/mL.
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Lower PMT setting on flow cytometry.