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Applications
Tips for panel design
1. Knowing the following features of your instrument is essential: spectral or conventional cytometer, available laser lines and arrangement, optical filters available as on the SH800.
2. Put the brightest fluorochromes on the dimmest markers or markers with variable expression. PE, for example is a bright fluorochrome and is a good option for lower antigen density targets.
3. Separate markers over as many lasers as possible to minimize spectral crosstalk. Where possible put co-expressing markers on different lasers.
4. Handle tandem dyes (such as PE-Cy7) with care by minimizing light exposure and time at room temperature. Include appropriate controls.
Example T-Cell, B-Cell, Natural Killer Cell Panel
| Specificity | Purpose |
| CD3 | T-cell lineage marker |
| CD4 | T-cell subset |
| CD8 | T-cell subset |
| CD19 | B-cell lineage |
| CD45 | Lymphocyte marker |
| CD56 | Natural killer cell (NK marker) |
How do we apply this to the example?
| Specificity | Purpose | Co‑Expression | Relative Expression | 2 Laser(Blue, Red) | 3 Laser(Blue, Red, Violet) | 4 Laser(Blue, Red, Violet, Yellow-Green) |
|---|---|---|---|---|---|---|
| CD3 | T-cell lineage marker | CD4, CD8 | High | APC | APC | APC |
| CD4 | T-cell subset | CD3 | High | FITC | FITC | FITC |
| CD8 | T-cell subset | CD3 | High | PE-Cy7 | PE-Dazzle 594 | PE-Dazzle 594 |
| CD19 | B-cell lineage | Medium | PE/Dazzle 594 | BV421 | BV421 | |
| CD45 | Lymphocyte marker | CD3, CD4, CD8, CD19, CD56 | High | PerCP-Cy5.5 | BV510 | BV510 |
| CD56 | Natural killer cell (NK marker) | Medium | PE | PE | PE |
