Hi Fei, thanks for the good word. Unstained beads will be used as control for compensation and unmixing if you are using the same beads for your positive group. The autofluorescence of both positive and negative must be the same. But for your FMOs, the question is different: you are trying to figure out the impact of all of your fluorophores (minus the one you're gating on) on the spread of the negative population. So the only appropriate way to create this control is to use cells.

@@UChicagoFlow thanks for your response. I am still confused with the unstained control. In my previous experience, I used unstained beads to adjust FSC and SSC to settle the cell population in the middle of plot. And set up the voltage of each color through the single control with beads for compensation. However, I didn’t do unstained cells to adjust that cell population. Beads could not the same size with the real cells. So, I want to know whether my design is acceptable through unstained beads to gate cell population instead of unstained cells? Sorry for my tedious words.

@@feitu6403 I typically set voltages on FSC and SSC specifically for the beads and cells. Most of the time, they have different properties, and will require you to decide on the settings to best showcase what you're actually looking at. As far as selecting the voltages for the fluorescent parameters detectors, I think your method likely puts you within range of what will allow you to separate your positive and negative markers without saturating the detectors. This is the whole point and if that is what you have right now, you are probably good to go. We go in more details about voltage selection in the next video in this series - here 13:05.

@@zxcv2677 thanks a lot!