ChromaTwist Dye to Antibody Conjugation and Multicolour Flow Cytometric Evaluation
Executive Summary
- The ChromaTwist dye (Gen-2-Dye) has been conjugated to the CD8 antibody (Ab) as shown by UV/Vis (Figure 1a) and emission spectroscopy (Figure 1b). In addition, the conjugate was shown to be active and bound to Protein A and G in the Abcam Conjugation Check kit (Figure 1c).
- The Gen-2-DyeCD8 conjugate when incubated with Anti-Mouse Ig, κ/Negative Control Compensation Particles Set in three different buffers lead to positive particle staining (BD Fortessa X-20 Flow Cytometer) (Figure 2) ranging from 21.2-44.5%. BUV395●CD8 was used as a commercial comparison in FACS staining buffer and gave 38.7 % +ve population compared to 31.1% for the Gen-2-Dye●CD8 conjugate. More technical details are set out below.
ChromaTwist Dye to CD8 Antibody Conjugation
A ChromaTwist dye (Gen-2-Dye) has been conjugated to CD8 RPA-T8 antibody (Ab). Characterisation of the Gen-2-Dye●CD8 conjugate (post Pierce™ Dye Removal Column and Amicon Ultra-0.5 (30 kDa) centrifugal filtration) to rigorously exclude free dye) with absorbance (Figure 1a) and emission (Figure 1b) spectroscopies together with a Conjugate Check Kit (Figure 2c) provided evidence of successful conjugation, and removal of free dye. Degree of labelling of upto 10 CT dyes per Ab was achieved.
![](https://chromatwist.com/wp-content/uploads/2022/12/Picture1-800x192.png)
Flow Cytometric Evaluation of the CT-Ab Conjugate
The headline result is that postive staining was achieved using the CT dye-Ab conjugate with Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (Figure 2). This staining was observed using the Indo-1 (blue) filter, which only detects part of the tail of the Gen-2-Dye●CD8 conjugate’s fluorescence (Figure 1b)). The staining revealed a buffer dependence with MES buffer affording a 21.2% positive stain, which increased with FACS (31.1%) and Tris HCl (44.5%) buffer. The BUV395●CD8 conjugate was used as a commercial comparison with FACS staining buffer and gave a positive stain population of 38.7%, relative to 31.1% with the Gen-2-Dye●CD8 conjugate. Conjugate aggregates were observed at room temp which inhibited partcle binding. Therefore, to reduce this aggregation bead staining was carried out at 37 °C, which reduced aggregate formation and enable particle binding, as shown in Figure 2.
![Figure 2. Flow cytometric evaluation of (BD Fortessa X-20) Gen-2-Dye●CD8 conjugate stained against compensation beads in three buffers.](https://chromatwist.com/wp-content/uploads/2022/12/Picture2-800x176.png)
Read more about ChromaTwist dye development for Spectral Flow Cytometry.