Lymph node cells lose TGN1412 reactivity by suspension culture. (A) Human lymph node (LN) cells were kept on ice or at 37°C (5% CO₂) for 1 hour in suspension (1 × 10⁶ cell/mL), and the proliferative response to TGN1412 (1 μg/mL) was measured by [³H]thymidine incorporation between stimulation day 2 and 3. One of 2 individual experiments with similar results is shown. (B) Fresh LN cells were placed in suspension in low cell density (1 × 10⁶ cell/mL), and either stimulated immediately (TGN1412 1 μg/mL) or kept in suspension culture at 37°C, 5% CO₂, for 2 or 4 hours (top panel). A PBMC sample from the same donor was analyzed in parallel (bottom panel). Cell proliferation was determined after 4 days of stimulation by Ki67 staining, gated on CD4⁺ cells. One of 2 individual experiments with similar results is shown.