Cytotoxicity of Anti-FCRL5 Immunotoxins on B and T Cells

The cytotoxic effects of the anti–FCRL-5 immunotoxins on purified B cells and T cells from HCV-infected patients with MC and healthy donors were measured by flow cytometry using 7-aminoactinomycin D (7-AAD) and annexin V staining. Prepared cell samples were seeded onto 96-well plates at 2 × 10⁵ cells/well. Serial dilutions of F56-IT and F25-IT in the culture medium were added to the cells, at a final concentration of 0–10,000 ng/ml. After 72 hours of culture, the cells were stained with PE–Cy7–conjugated anti-CD19 or Alexa Fluor 700–conjugated anti-CD3 and FITC–conjugated anti-CD45 (Beckman Coulter), and then resuspended in annexin binding buffer and labeled with 7-AAD and PE-conjugated annexin V (BD PharMingen). Flow cytometric analyses were performed on a Navios flow cytometer, with results analyzed using Navios software (Beckman Coulter). Viability was assessed by setting the gates based on the light-scatter properties of the lymphocytes. Viable cells were those negative for 7-AAD and PE-conjugated annexin V. To compensate for spontaneous cell death, the relative percentage of viable cells at the end of the assay was calculated using the following formula: (no. of CD19+ or CD3+ viable cells recovered in the treated well/no. of CD19+ or CD3+ viable cells in the untreated well) × 100 (41 (link)). All experiments were performed in duplicate.