Table 2.
Potential multicolor panels for Flow-FISH using previously validated antibodies/fluorochromes and BS3 cross-linkinga
| Fluorochrome | Target/Antigen (reference) | Company | Excitation (nm) (laser) | Emission |
|---|---|---|---|---|
| Panel 1 | ||||
| Hoechst 33342 (HO342) | DNA stain (Schmid, Dagarag et al. 2002) | Life Technologies | 361 (355) | 497 |
| Alexa Fluor 488 | CD8 (Schmid, Dagarag et al. 2002) or CD28 (Schmid, Dagarag et al. 2002) | BD Biosciences | 494 (488) | 518 |
| PE or Alexa Fluor 546 | CD57 PE (Baerlocher, Vulto et al. 2006) – used after FISH or CD8 labeled with GAM Alexa Fluor 546 (Schmid, Dagarag et al. 2002) |
BD Biosciences BD Biosciences/Life Technologies (GAM Fab’2 Alexa Fluor 546) |
496 (488, 561) 556 (488, 561) |
578 573 |
| Qdot 605 | CD14 (Kapoor, Hakim et al. 2009) | Thermo Scientific | Broad UV and violet excitation (350, 405) | 605 |
| Cy5 | PNA probe (Schmid, Dagarag et al. 2002) | Panagene | 647 (633) | 665 |
| Qdot 705 | CD4 (Kapoor, Hakim et al. 2009) | Thermo Scientific | Broad UV and violet excitation (350, 405) | 705 |
| Panel 2 | ||||
| Alexa Fluor 488/FITC | PNA probe (Kapoor, Hakim et al. 2009) | Dako, Panagene | 494 (488) | 518 |
| Alexa Fluor 546 | CD8 (Schmid, Dagarag et al. 2002) | BD Biosciences/Life Technologies (GAM Fab’2 Alexa Fluor 546) | 556 (561b) | 573 |
| Qdot 605 | CD14 (Kapoor, Hakim et al. 2009) | Thermo Scientific | Broad UV and violet excitation (350, 405) | 605 |
| Qdot 655 | CD3 (Kapoor, Hakim et al. 2009) | Thermo Scientific | Broad UV and violet excitation (350, 405) | 655 |
| Qdot 705 | CD4 (Kapoor, Hakim et al. 2009) | Thermo Scientific | Broad UV and violet excitation (350, 405) | 705 |
| LDS-751 | DNA stain (Baerlocher, Vulto et al. 2006, Kapoor, Hakim et al. 2009) | Thermo Scientific | 543 (488, 561, 532) | 712 |
a
Note that successful implementation of these expanded panels that extend beyond the reagent combinations that have been published, i.e., by Schmid et al. (Schmid, Dagarag et al. 2002) and Kapoor et al. (Kapoor, Hakim et al. 2009), will depend on the instrument configuration, laser options, and filter sets and will require careful establishment of instrument settings and fluorescence compensation.
b
Although Alexa Fluor 546 can also be excited by a 488nm laser (see first panel), for the second panel, exciting it with a 561nm laser is preferable.