flow cytometry experiments

How do you properly set unstained and single stained controls for flow cytometry experiments?

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In the case of PI + Annexin V, where there are no antibodies involved, do I still need single stain controls? Measurement would be using 2 spatially separated lasers, so there will be no need for compensation.
3. If I am using 3 different lasers for the PI+ Edu + pHH3, do I still need single stain controls?
Just for clarity, how many tubes will I need to prepare for my experiment?
1. untreated – unstained
2. untreated – PI
3. untreated – Edu
4. untreated – pHH3
5. untreated – PI + Edu + pHH3

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