Apc anti human cd33 antibody

Human dental pulp stem cells (DPSC) were obtained from Lonza (PT-5025). The stemness of human dental pulp stem cells (DPSCs) (p. 16) was confirmed by flow cytometry as previously described [30 (link)]. The expression of positive (CD105) and negative (CD33) markers of mesenchymal stem cells was analyzed, using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, cells were stained with PE anti-human CD105 antibody (323205, Biolegend, San Diego, CA, USA) and APC anti-human CD33 antibody (303407, Biolegend, San Diego, CA, USA), following the manufacturer’s instructions.

We performed the cytotoxicity assays by co-culturing engineered TILs with primary AML blasts from the same patient (isolated by CD33-microbeads pull-down) in 24-well plates. AML blasts from BMMNC were separated using APC anti-human CD33 antibody (Biolegend), anti-APC microbeads (Miltenyi Biotech, Germany), and a MiniMACS™ Separator with an MS Column. The ratio of autologous TILs to AML blasts were in the range of 5:1–10:1 according to a previous report [20] (link). After overnight incubation, cells were collected, stained, and processed for FACS assay of biomarkers including viability dyes (Invitrogen™) and CD33 according to manufacturers’ protocols. Analyses and graphs will be generated using the GraphPad Prism software to evaluate significance.

In vitro targeting of anti-CD33 Nbs was evaluated on THP-1 cells, using flow cytometry. THP-1 cells (2 × 10⁵/100 µL) were preincubated, with or without 1 µg of each anti-CD33 Nb and the non-targeting control Nb, for 1 h at 4 °C. After washing cells with cold PBS, cells were incubated with 150 ng of FITC-anti-HA.11 Ab (BioLegend, San Diego, CA, USA), at 4 °C, for 30 min. In another condition, THP-1 cells incubated only with 200 ng of APC anti-human CD33 antibody (BioLegend) were used as a positive control. Cells incubated with 200 ng of APC anti-Mouse IgG1 antibody κ (BioLegend) were used for the isotype control. Flow cytometry was performed on a FACS CANTO II analyzer, and data were processed by using FlowJo Software (BD Biosciences, San Jose, CA, USA).