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Technical Note Executive Summary: Technical Note Executive Summary: Reported here is the characterisation of the blue and the green �Ǹ�Թ� (CT) Dye CD4 (mouse anti-human IgG1, Clone: SK3) conjugates (Figure a-c). Flow cytometry was performed on UltraComp eBeads™ compensation beads with fresh and aged conjugates with conjugates stored at 4 °C, in the dark in between measurements. The staining indices (Figure d) are enhanced over those reported previously (Technical Note 5), data is summarised as follows:
Conclusion: Significant improvement of staining indices since Technical Note 5 (May 2023) and the conjugates are photostable over a period of at least a week (stored in the refrigerator 4 °C; stability testing is on-going).

CT Dye●C��4 Conjugate Detection using a Conjugate Check Kit, Figure a: Successful conjugation is evident from the fluorescence observed at the “test line” upon irradiation with 302 nm light, indicating that the CD4 antibody was indeed labelled with the �Ǹ�Թ� dyes. The free dye controls (samples without antibody) show no staining at the test line.

Comparison of CT Dye●C��4 Conjugate Absorption Spectra, Figure b: The absorption spectra of CT450●C��4 and CT525●C��4 are compared to the unconjugated antibody, revealing the conjugate has an absorption profile containing elements of the dye and antibody, and a significant shift in the antibody λ_max (20 nm) giving good evidence of conjugation. The absorption spectra of conjugates show that they are excited by the conventional 355 nm laser used in flow cytometry, but also the 320 nm laser system recently developed by Sony Biotechnology in their ID7000 instrument.

CT Dye●C��4 Conjugate Emission Spectra, Figure c: The emission spectra of CT450●C��4 and CT525●C��4 conjugates show peak emission is observed at 465 nm and 479 nm respectively, which is congruent with the free blue and 525 emission profiles.

UltraComp eBeads™ Staining with CT Dye●C��4 Conjugate and Flow Cytometry, Figure d:��Stained UltraComp eBeads™ were analysed on a LSR Fortessa X-20. Beads were gated to exclude debris and doublets and fluorescence is observed in the indo-1 (violet) and indo-1 (blue) fluorescence channels for the 1 day old blue and green CT450●C��4 and CT525●C��4 conjugates, respectively, affording staining indices of 53 and 38. The CT450●C��4 and CT525●C��4 conjugates were tested again after 9 and 13 days respectively, affording staining indices of 49 and 43, indicating stability over at least a week in (on-going) stability tests.

�Ǹ�Թ� is currently working on a development program to improve dye conjugate purification, optimise Degree of Labelling (dye/protein ratio) and perform cell staining. We are also developing technology to push these staining indices up by factors of 2, 3, 4, 6, 8, and 9 and potentially beyond via a scalable amplification technology and expand our conjugated dye portfolio to include red emissive dyes.

Conjugate Check Kit Experimental:

Solutions of the CT dye●C��4 conjugates (5 μL of conjugate, 35 μL 1x running buffer/1 % BSA) were run on a Conjugation Check Kit (Abcam ab236554).

��Staining Beads and Flow Cytometry Experimental:

1. Vortex (20 s) and add compensation beads (Invitrogen 01-2222-42, 25 µL) to a 1.5 mL Eppendorf.
2. Add �Ǹ�Թ� dye●C��4 antibody conjugate (0.5 µL) to the beads, ensuring the conjugate is deposited directly in to the beads.
3. Incubate the sample in the dark (30 min, 21 °C).
4. Add staining buffer (Biolegend cat no. 420201, 1400 µL) and vortex (3 s).
5. Centrifuge to wash (600 g, 5 min).
6. Remove supernatant, leaving behind the bead pellet.
7. Resuspend the bead pellet in staining buffer (1400 µl), vortex (3 s) and centrifuge (600 g, 5 min) – repeat steps ⑥ and ⑦ for a total of three washes.
8. Resuspend the bead pellet in 500 µL staining buffer, vortex (3 s) and transfer the sample to a FACS tube.
9. Samples are analysed on a LSR Fortessa X-20 flow cytometer. Beads are gated to exclude debris and doublets and fluorescence is observed in the indo-1 (Violet) ≡ 450/50 and indo-1 (Blue) ≡ 525/50 fluorescence channels for the CT450●C��4 and CT525●C��4 conjugates, respectively.