RAW 264.7 cells (0.05 × 106/0.5 mL/well) and THP-1, MC38, and HT-29 cells (0.1 × 106/0.5 mL/well) were seeded in 48-well plates (Corning Costar, Corning, NY, USA). The next day, FITC-labeled BN-17 at concentrations of 1, 50, and 100 µg/mL was added. After 4 and 24 h of incubation with the compound, the cells were collected and suspended in PBS supplemented with 2.5% FBS (Biowest, Nuaillé, France). The mean fluorescence intensity (MFI) as well as changes in the size and granularity of cells were analyzed based on forward scatter (FSC) versus side scatter (SSC) using a FACS Fortessa instrument with Diva software version 8.0 (Becton Dickinson). DAPI dye (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was added to each tube to eliminate dead cells. Histograms and density dot plots were prepared using NovoExpress 1.3.0 software (Agilent Technologies, Inc., Santa Clara, CA, USA).
Diva software version 8
Manufactured by BD
Diva software version 8.0.1 is a laboratory analysis software that enables data processing and visualization for various laboratory instruments and experiments. It provides a platform for managing and analyzing data generated from these devices.
Automatically generated - may contain errors
Lab products found in correlation
Rnalater, by Thermo Fisher Scientific (2 mentions)
48 well plate, by Corning (1 mentions)
Dapi dye, by Thermo Fisher Scientific (1 mentions)
Novoexpress 1, by Agilent Technologies (1 mentions)
Facs fortessa, by BD (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dnase 1, by Roche (1 mentions)
Facsaria 2, by BD (1 mentions)
Tcr vβ repertoire kit, by Beckman Coulter (1 mentions)
Accucount, by Spherotech (1 mentions)
7 aad, by BioLegend (1 mentions)
Pharm lyse, by BD (1 mentions)
5 protocols using diva software version 8
1
Cellular Uptake of FITC-labeled BN-17
2
Isolation and Characterization of Bone Marrow Cells
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Mononuclear cells of bone marrow aspirates were thawed and then resuspended in RPMI medium containing DNAse I (Roche), followed by a wash with cold 0.1% bovine serum albumin (BSA, Sigma) in phosphate buffered saline (PBS). Cells were then stained with the following antibodies (Supplementary Table 2 ): CD34, CD45, GD2 (kindly provided by Prof. Handgretinger, University of Tübingen), and L1CAM (CD171) in PBS/BSA 0.1% for 10 min at 4 °C. Finally, cells were stained with DAPI (Sigma) for 5 min at 4 °C and were passed through a 70 µm cell strainer into FACS tubes in PBS/BSA 0.1%. Acquisition and sorting of live cells were performed with FACSAria and the DIVA Software version 8.0 (Becton Dickinson). Sorted single cells were split into halves and were taken forward for scATAC-seq and scRNA-seq processing.
3
Unbiased TCR Profiling of EBV-Specific CD4+ T Cells
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Viable EBV-specific tetramer+ CD4+ T cell populations were sorted at >98% purity directly into RNAlater (Thermo Fisher Scientific) using a custom-modified FACSAria II flow cytometer equipped with DIVA software version 8.0.1 (BD Biosciences). Unbiased amplification of all expressed TRB gene rearrangements was conducted using a template-switch–anchored RT-PCR with a 3′ C region primer (31 ). Amplicons were subcloned, sampled, sequenced, and analyzed as described previously (32 (link)). Gene use was assigned using the International ImMunoGeneTics nomenclature (33 (link)). All functional TCR sequences were deposited online at VDJdb (34 (link)). Expression of defined TCR Vβ segments on the surface of EBV-specific CD4+ T cells was assessed using a TCR Vβ Repertoire Kit (Beckman Coulter).
4
Comprehensive Leukocyte Immunophenotyping
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Using reverse pipetting, 100 μl of whole blood collected on EDTA-coated tubes were incubated with anti-CD32 to prevent FC-mediated non-specific binding according to the manufacturer’s instructions. The panel of antibodies as well as the gating strategy were inspired by Barnett-Vanes and al. [20 (link)] and detailed in the S1 Table . Briefly, a panel of 11 antibodies was used to identify the neutrophils (CD45+/ SSChi / His48+), the monocytes (CD45+/ SSClo/ His48hi or lo/CD43hi or lo), the B lymphocytes (CD45+/ SSClo /CD45R-B220+), the T lymphocytes (CD45+/ SSClo / CD3+/CD4+ or CD8+), and the natural killer cells (CD45+/SSClo /CD161a+). The whole blood samples were incubated with the antibodies for 20 minutes at room temperature and the BD Pharm Lyse solution was then added for 10 minutes to lyse the erythrocytes. Samples were stored at 4°C until analysis by flow cytometry (LSRII BD Biosciences). Accucount (Spherotech) were added to establish a cell count per μl of blood. Data were analyzed on Diva software version 8.0.1 (BD Biosciences).
5
Quantification of Immune Cell Populations
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Whole blood samples collected on EDTA-coated tubes were incubated or not with LPS (500 ng/mL) for 30 minutes at 37°C. The samples were then stained using mouse anti-rat CD45 Alexa Fluor 700 (clone OX-1, conjugated to Alexa Fluor 700, Biolegend), mouse anti-rat CD11b antibody (clone WT.5, conjugated to V450, BD Horizon), and 7AAD (Biolegend) for 30 minutes at 4°C. FACS lysing solution (BD Pharm Lyse, BD Biosciences) was added for 15 minutes at room temperature and samples were stored at 4°C until analysis by flow cytometry (LSRII, BD Biosciences). Data were analyzed on Diva software version 8.0.1 (BD Biosciences).
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