Facscalibur 2 cytometer

Wells were first coated with 2 μg/ml of anti-CD3 mAb for 24 h at 4°C, after which, they were washed with PBS. The wells with or without anti-CD3 were then coated with 2 μg/ml collagen for 3 h at 37°C. After three washes with PBS, we seeded the cells for 3 h, and treated them with 10 μM of MTX for 24 h at 37°C. Cell apoptosis was determined using annexin V-FITC staining and measured by flow cytometry (BD FACSCALIBUR II cytometer).

The cells were labeled with conjugated control or with specific antibodies against integrins and CD5 for 40 min at 4 °C. For ABCC1 expression, we first permeabilized and fixed the cells with a cytoFix/CytoPerm kit (BD Biosciences) for 30 min at 4 °C, after which, we incubated them with FITC-conjugated isotypic control or anti-ABCC1 antibodies. After antibody staining, the cells were washed and analyzed for integrin, CD5 and ABCC1 expression levels by flow cytometry using BD FACSCalibur II cytometer, and a Canto II flow cytometer (BD Biosciences) for the analysis of β1 integrin on T-ALL blasts.

All phenotypic studies were performed on fresh whole blood using a four-colour BD FACScalibur II cytometer (BD, Paris, France). Flow cytometry data were acquired and collected by BD CellQuest Pro^TM and were analysed using FlowJo version 5.0. Lymphocytes were gated in the FSC/SSC dot plot, and NK cells were identified as CD3 negative and CD56 positive, and/or CD16 positive lymphocytes. NK-cell subsets were identified by co-expression of CD56 and CD16 on NK cells: CD56^brightCD16^neg (CD56⁺⁺CD16⁻) NK cell, CD56^dimCD16^neg (CD56⁺CD16⁻), CD56^dimCD16^pos (CD56⁺CD16⁺), and CD56^negCD16^pos (CD56⁻CD16⁺).
NK-cell natural cytotoxic receptors: NKp30 (CD337), NKp44 (CD336), NKp46 (CD335), and NK-cell Ig-like receptors (KIR): KIR2DL1/KIR2DS1(CD158a/h), and KIR2DS4 (CD158i), CD160, NKR-P1A (CD161), NKG2A (CD159a), NKG2C(CD159c), NKG2D (CD314), DNAM-1 (CD226), IL-2Rβ (CD122), 2B4 (CD244), and the activation marker CD69 were detected using monoclonal antibodies from Beckman Coulter (Paris, France), RD Systems (Paris, France) and BD Biosciences (Paris, France). The list and the combination of fluorochrome-conjugated monoclonal antibodies used for immune-phenotyping are shown in Supplementary Table S1. The gating strategy of NK cells is shown in the Supplementary Figure S1.

The cells were stained with (PE-conjugated) isotypic control, anti-α2 integrin, and anti-ABCG2 antibodies for 30 min on ice. For ABCC1 expression, we first permeabilized and fixed the cells with a cytoFix/CytoPerm kit (BD Biosciences) for 20 min, after which, we incubated them with FITC-conjugated isotypic control or anti-ABCC1 antibodies. The cells were washed and then analyzed for α2 integrin, ABCC1, and ABCG2 expression levels by flow cytometry (BD FACSCalibur II cytometer).

All antibodies and isotype controls for cell labeling were purchased from eBioscience (USA) and were diluted in PBS with 2% normal mouse serum (Sigma-Aldrich). After 132-hour coculture incubation, the cells were stained in a total volume of 50 μL with anti-CD4-FITC (clone RM4-5, final concentration 1.2 μg/mL) and anti-CD25-PE (clone PC61.5, 0.24 μg/mL) or isotype controls which were used at the same concentration as specific antibodies. After 45 min of incubation at 4°C, the cells were washed twice in PBS and then fixed in 1% buffered formaldehyde. The samples were acquired with a FACSCalibur II Cytometer (Becton, Dickinson and Company). We performed FACS analysis to determine the ratio of total number of activated (CD4⁺CD25⁺) T cells to nonactivated (CD4⁺CD25⁻) cells.

To detach cells from the culture dishes, Accutase Cell Detachment Solution (Becton Dickinson) was used. Centrifuged cells (1 × 106) required for the analysis were suspended in a cold stain buffer (Becton Dickinson). Using Human MSC Analysis Kit (Becton Dickinson), according to manufacturer's protocol, to each tube was added the appropriate dilution of fluorochrome-conjugated antibodies directed against APC CD73, FITC CD90, and PerCP-Cy™5.5 CD105 (positive markers) and PE CD34, PE CD11b, PE CD19, PE CD45, and PE HLA-DR (negative markers) and incubated for 30 minutes at room temperature, protected from light. After incubation, cells were washed twice with stain buffer, suspended in 500 μl of this buffer and immediately analyzed using FACSCalibur II cytometer (Becton Dickinson). 10,000 events were counted using FACSDiva software computer program.