Extended Data Fig. 2. The quantitative selectivity with which cells take up NTUs.

(a) Gating strategies of five different cell types. Hoechst 33342⁺ DiI^- DiD⁺ cells were defined as nucleus pulposus cells (NPCs). Hoechst 33342⁺ DiI⁺ DiD^- cells were defined as chondrocytes. Hoechst 33342⁺ DiI^- DiD^- cells were defined as fibroblasts. Hoechst 33342^- DiI⁺ DiD^- cells were defined as muscle satellite cells (SCs). Hoechst 33342^- DiI^- DiD⁺ cells were defined as macrophages. The following results indicate the proportions of the five types of cells that took up CM-NTUs: 69.9% (chondrocytes), 0.9% (NPCs), 2.2% (fibroblasts), 8.8% (SCs), and 9.4% (macrophages). Based on the level of mean fluorescence intensity (MFI) and using 5.8 × 10⁴ NTUs per chondrocyte as the standard, we estimated that the numbers of NTUs taken up by NPCs, fibroblasts, SCs, and macrophages were ~6.4 × 10³, 8.6 × 10³, 1.8 × 10⁴, and 1.6 × 10⁴ per cell, respectively. (b) Gating strategies of chondrocytes. Scheme #1 (left panel): Hoechst 33342⁺ DiO⁺ cells were defined as chondrocytes taking up CM-NTUs. Hoechst 33342⁺ DiI⁺ cells were defined as chondrocytes taking up NPC membrane-NTUs (NPCM-NTUs). Hoechst 33342⁺ DiD⁺ cells were defined as chondrocytes taking up macrophage membrane-NTUs (MM-NTUs). Scheme #2 (right panel): Hoechst 33342⁺ DiO⁺ cells were defined as chondrocytes taking up CM-NTUs. Hoechst 33342⁺ DiI⁺ cells were defined as chondrocytes taking up SC membrane-NTUs (SCM-NTUs). Hoechst 33342⁺ DiD⁺ cells were defined as chondrocytes taking up fibroblast membrane-NTUs (FM-NTUs). The results showed that chondrocytes took up the highest percentage of CM-NTUs, at 67.6% and 58.7% in the two experiments, respectively. The proportions of chondrocytes that took up the NPCM-NTUs, MM-NTUs, SCM-NTUs, and FM-NTUs were 3.2%, 1.0%, 9.9%, and 8.6%, respectively. Based on the MFI, we estimated the number of NPCM-NTUs, MM-NTUs, SCM-NTUs, and FM-NTUs taken up per chondrocyte was ~4.8 × 10³, 2.9 × 10³, 3.2 × 10⁴, and 1.7 × 10⁴, respectively.