You will need to bring an unstained control, single colour compensation controls and you MAY need to bring FMO (Fluorescence Minus One) gating controls. You may want to consider an FMO control if you are using more than 5 colours simultaneously and if your populations of interest cannot be discretely separated from background/negatives.
Please refer to the table below to see an example of a typical sort experiment with three colours.
| Control type | Sample Name | Marker + fluorochrome #1 | Marker + fluorochrome #2 | Marker + fluorochrome #3 |
|---|---|---|---|---|
| Unstained Control | Unstained | - | - | - |
| Single colour compensation controls | CD3 - FITC | CD3 - FITC | - | - |
| CD4 - PE | - | CD4 - PE | - | |
| CD8 - APC | - | CD8 - APC | ||
| FMO Control | FMO FITC | - | CD4 - PE | CD8 - APC |
Fluorophores excited by the Yellow/Green laser
If you are using markers such as mCherry, RFP, Tomato Red, Texas-Red, etc, Please contact the Flow Facility to inquire about the specific sorter to book as only a limited number of sorters are equipped with an appropriate laser to detect these signals.
Viability Dyes
We recommend that you add a viability dye such as PI, 7AAD, or LIVE/DEAD fixable dyes (Invitrogen) to your sample to enable exclusion of dead cells during sorting. If you choose to do so, please bring an appropriate single color compensation control. We suggest the following: Heat killed cells (65oC for 15 minutes and then mix with live cells) followed by staining with viability dye.