Anti human cd19

After harvesting, B-ALL cells or HUVECs (0.1-1x10⁶) were centrifuged at 500 g for 5 min at room temperature and resuspended in 100 µl PBS containing anti-human CD19 (#302206, 1:20 dilution, Biolegend, Inc.), anti-human CD49d (#304304, 1:20 dilution, Biolegend, Inc.) and anti-human CD29 (#303008, 1:20 dilution, Biolegend, Inc.) antibodies, alongside the isotype control mouse IgG1κ (Biolegend, Inc.). Following incubation at 4˚C for 30 min, cells were washed with 1 ml PBS and resuspended in 100 µl DAPI/PBS (0.1µg/ml) for 15 min at 4˚C for assessment using a flow cytometer (BD FACSCanto II, BD Biosciences). Flow cytometry data were analyzed from the viable cells in the DAPI-negative population, based on an isotype gating strategy using Flow Jo (version 10, BD Biosciences).
For apoptosis assay, B-ALL cells were treated for 48 h with either DMSO or AVA4746 25 µM, with or without VDL chemotherapy (vincristine 5 nM, dexamethasone 0.05 nM, L-asparaginase 2.5x10^-3 IU/ml) in the presence or absence of OP9. Annexin V (#640947, 1:20; Biolegend, Inc.) and DAPI were used to stain the 1x10⁶ cells for 15 min at room temperature (25˚C), in the dark.

Primary PCNSL cells were collected and fixed in cold 2% paraformaldehyde for 15 min. The cells were then washed thrice with cold PBS. For CD19 and CD20 expression analysis, cells were blocked in Human Trustain FcX (BioLegend, #422302) at 37°C for 30 min and divided into blank, isotype, and detection tubes. Cells in the isotype tube were incubated with CD19 (Biolegend, #400161) and CD20 isotype antibodies (Biolegend, #400149), followed by incubation with anti-human CD19 (Biolegend, #302244) and anti-human CD20 (Biolegend, #302326) antibodies at room temperature for 30 min. Finally, cells were washed twice and resuspended in cold PBS. All the analyses were performed using Flowjo software (version 10.8.1, BD).