Data Requirements
The performance of the 1-click analysis depends on the quality of the controls used to unmix your experimental samples.
Panel design
- A well-designed panel that accounts for autofluorescence properties, material types, and spectral similarity
Single-stain controls
- At least one control for each fluor in your panel.
- Each control used to unmix experimental files must be high-quality. They should:
- At minimum contain 100 positive events in each control.
- Stain at least as brightly as your brightest multicolor experimental files.
- Stain within the dynamic range of the instrument.
- Emit spectral signatures that are representative of their performance in your experimental samples.
Unstained controls
Provide unstained controls at the highest level you can.
| Preference | What to provide | When to use it |
|---|---|---|
| Best | One unstained control paired with each multicolor file | Whenever possible |
| Next best | One unstained control for each condition in every run | If paired controls aren’t feasible |
| Minimum | One unstained control for each condition (sample type × preparation method), set as the “source of background” for the matching fully stained files | If per-run controls aren’t feasible |
| As controls move from Best to Minimum categorization, the chance of generating sub-optimal outputs to support the 1-click analysis increases. | ||
Cytometer settings
- Ensure that cytometer settings (e.g. voltages) are comparable between the control files you use to unmix your multicolor experimental files and the multicolor experimental files themselves.
- Ensure all populations you intend to measure with your panel are on-scale across all detectors on your device.
Sample matrices
- 1-click analysis was primarily tested against PBMC samples during its development. While other sample matrices can be processed through the 1-click system, if the panel does not account for their autofluorescence properties, and unstained material for those matrices is not available, the chance of generating sub-optimal outputs to support the 1-click analysis increases.