Table 3.
Recommended Assay and Instrument Controls for Measuring Immune Cell Proliferation
| Cells | Label | Treatment | Notes | |
|---|---|---|---|---|
| Assay controlsa | Teff | CFSE | +acc, no stimulusd | Negative control: spontaneous Teff proliferation |
| Teff | CFSE | +acc + stimulus | Positive control: maximum Teff proliferation | |
| Treg | Claret | +acc, no stimulus | Negative control: spontaneous T regulatory proliferation | |
| Treg | Claret | +acc + stimulus | Define Treg proliferation | |
| Teff | CFSE | PHA | Positive control: verify that Teff are able to proliferate when cell surface receptors are bypassed | |
| acc | None | None | Optional: confirm that they remain CD4− throughout assay and will not be confused with highly divided Teff | |
| Teff | Varying CFSE | +acc + stimulus | Staining control: used during assay setup to verify that concentration of tracking dye chosen does not affect Teff proliferation (Fig. 1c)e | |
| Treg | None | +acc + stimulus | Staining control: used during assay setup to verify that tracking dye labeled cells proliferate equivalently to unlabeled cells (Fig. 9)e | |
| Instrument controlsb | Teff | None | Unstimulated, accessory only | Set voltage and negative region in CFSE channel |
| Teff | CFSE | Unstimulated | Set voltage in CFSE channel; set color compensation in other channels; estimate location of undivided Teff | |
| Treg | None | Unstimulated | Set voltage and negative region in Claret channel | |
| Treg | Claret | Unstimulated | Set voltage in Claret channel; set color compensation in other channels; estimate location of undivided Treg | |
| Teff (no Claret) | CD4 PE-Cy7 | Unstimulated | Set compensation | |
| acc + Teff (no CFSE)c | LIVE/DEAD Fixable Violet | Unstimulated | Set compensation | |
a
Assay controls are included in the experimental plate with test samples to verify that the expected biological outcomes can be recognized using the chosen instrument conditions
b
Instrument controls are used to establish instrument voltages and settings
c
Irradiated accessory cells will be nonviable and should be 100% positive for this viability probe
d
Acc = accessory cells (CD4 negative lymphocytes); stimulus = anti-CD3 plus anti-CD28
e
Needed only to establish optimized staining conditions for tracking dye when assay is first being implemented in the laboratory; not required on a routine basis