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Lifetime Flow Cytometry
SETA BioMedicals has developed some proprietary fluorescent dyes, tandems, and labels (fluorochromes) optimized to enable robust, fluorescence lifetime (FLT)-based, single- and multiple-cell analyses with Single Cell Fluorescence Lifetime Flow Cytometry (SC-FLFC) or High-Throughput Fluorescence Lifetime Imaging Flow Cytometry (HTFLIM-FC) [1]. These FLT-based reporters span the full spectral range from UV to near-IR, with luminescent lifetimes ranging from ps to tens of ns and are compatible with the Danube Fluorescence Lifetime Flow Cytometer (Kinetic River Corp., USA) [2], Swabian Instrument FLFC [3], and other non-commercial FLT and FLIM FCs.
| Excitation laser line [nm] |
Free dye / Ab conjugate‡ in phosphate buffer pH 7.4 |
Environment sensitive FLT |
λmax Ab [nm] |
λmax Fl [nm] |
Brightness at the excitation laser line [M–1cm–1] |
FLT† [ns] |
|
380 |
Seta-375 / IgG (D/P = 5) |
No |
372 / 372 |
476 / 480 |
20,500 / 50,000 |
3.2 / 3.5 |
|
SeTau-380 / BSA |
No |
381 / 383 |
480 / 468 |
900 / 3,000 |
32.5 / 30.6 |
|
|
405 |
SeTau-405 / IgG (D/P = 2) |
No |
405 / 405 |
518 / 518 |
11,000 / 6,900 |
9.3 / 8.9 |
|
436 |
SeTau-425 / BSA (D/P = 2) |
n.d.* |
425 / 427 |
545 /532 |
1,500 / 1,000 |
26.2 / n.d.* |
|
Seta-470 / Glycerol:Water 10 cP, +65 °C / 700 cP, -6 °C |
Yes, Viscosity |
460 / 460 / 460 |
521 / 525 / 525 |
560 / n.d.*/ n.d.* |
0.21 / 1.7 / 17.3 |
|
|
488 |
SeTau-488 / IgG (D/P = 2) |
Yes |
486 / 484 |
532 / 530 |
22,800 / 71,600 |
1.2 / 2.3 |
|
SeTau-500 / IgG (D/P = 2) |
Yes |
502 / 500 |
568 / 563 |
31,200 / 53,100 |
1.7 / 2.6 |
|
|
Seta-470 / Glycerol:Water 10 cP, +65 °C / 700 cP, -6 °C |
Yes, Viscosity |
460 / 460 / 460 |
521 / 525 / 525 |
560 / n.d.*/ n.d.* |
0.21 / 1.7 / 17.3 |
|
|
532 |
Seta-555 / (D/P = 2) |
No |
555/ 556 |
570/ 571 |
3,400 / n.d.* |
0.2 / n.d.* |
|
594 |
Seta-633 / IgG (D/P = 2) |
Yes |
633 /637 |
644 /647 |
3,600 / 23,700 |
0.25 / 1.49 |
|
Seta-640 / IgG (D/P = 2) |
Yes |
636 / 648 |
646 / 656 |
4,600 / 19,900 |
0.7 / 2.7 |
|
|
635 |
No |
647 |
665 |
50,300 |
1.0 |
|
|
Seta-660 / IgG (D/P = 2) |
n.d.* |
661 / 665 |
672 / 675 |
4,900 / 38,900 |
1.5 / n.d.* |
|
|
SeTau-647 / IgG (D/P = 2) |
No |
649 / 648 |
695 / 694 |
74,000 / 128,600 |
3.2 / 3.1 |
|
|
650 |
No |
651 |
671 |
65,400 |
1.0 |
|
|
Seta-660 / IgG (D/P = 2) |
n.d.* |
661 / 665 |
672 / 675 |
10,700 / 83,800 |
1.5 / n.d.* |
|
|
SeTau-665 / IgG (D/P = 2) |
No |
664 / 662 |
712 / 716 |
47,900 / 54,200 |
3.1 / n.d.* |
|
|
SeTau-670 / IgG (D/P = 4) |
No |
673 / 672 |
694 / 693 |
39,600 /61,600 |
1.62 / 1.75 |
|
|
Square 650-pH at pH 5.6 / pH 9.0 |
Yes, pH§ |
653 / 535 |
671 / 663 |
20,900 / 260 (3,600 at λ* = 532 nm) |
1.2 / 0.5 |
|
|
Square-660/Oligonucleotides |
Yes |
657/676 |
676/695 |
n.d.* / n.d.* |
0.29/2.73 |
|
|
680 |
No |
676 |
694 |
50,400 |
1.0 |
|
|
No |
683 |
705 |
122,300 |
2.9 |
||
|
Seta-670 / IgG (D/P = 5) / BSA (D/P = 1.5) |
Yes |
667 / 670 / 681 |
688 / 693 / 696 |
8,200 / 11,700 / 47,300 |
0.42 / 0.85 / 2.43 |
|
|
700 |
No |
749 |
774 |
8,400 |
0.57 |
|
|
750 |
Seta-750 / IgG (D/P = 2) |
No |
753 / 756 |
780 / 783 |
31,600 / 68,700 |
0.6 / n.d.* |
|
780 |
No |
777 |
807 |
2,600 |
0.6 |
† Acquired with ISS Chronos FD at 25 °C
‡ Fluorochrome conjugated to antibody (protein). D/P is dye-to-protein ratio
* Not determined
§ pH-sensitive dye, excitable at 650 nm under acidic pH and at 532 nm at basic pH, emitting at 660–670 nm under all conditions [4].
References
[1] Nat Commun 15, 7376 (2024).
[2] https://www.kineticriver.com/wp-content/uploads/2022/05/KRC-Danube-Data-Sheet.pdf
[3] https://www.swabianinstruments.com/applications/fluorescence-lifetime-flow-cytometry/
[4] Analytical Biochemistry, 2009, V390, 136-140.
