Penicillin

B16-F10, 4T1, Mel-Rel, TS/A, MCF-7, MDA-MB-468, LAU 145, and Me290 cell lines were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO₂. Cells were grown until 75% confluency and passaged using trypsin/1× EDTA (Eurobio), washed with PBS, and resuspended in supplemented RPMI-1640.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. CD19+ CD5+ B-cells were isolated from PBMCs using magnetic-bead-activated cell sorting, using a B-CLL cell isolation kit (Miltenyi Biotec). To evaluate the effect of AA on normal lymphocytes, naïve B-cells were isolated from donor lymphocyte infusions using a Naïve B-cell Isolation kit (Miltenyi Biotech). The purity assessed by CD19 expression on flow cytometry was around 98%. OSU-CLL cells were a gift from E. Hertlein and colleagues [28 (link)]. JVM3 cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Freshly isolated B-cells and CLL cell lines were cultured in RPMI 1640 medium (PAN Biotech, #P04–16500) with 10% fetal bovine serum (FBS) (PAN Biotech, #P30–3306) and L-glutamine, penicillin and streptomycin (1%) (Eurobio Scientific). When indicated, CLL B-cells were cultured in Iscove’s modified Dulbecco’s Medium (IMDM) (Merck, #FG0465) and alpha-MEM medium (Sigma Aldrich, #M4526) (n = 7). Cells were cultured at a density of 4 × 10⁵/ml in 48-well plates and were treated with either vehicle or AA or drugs for 24 h. To simulate hypoxia condition, cells were cultured in presence of 100 μM Cobalt(II) chloride hexahydrate (CoCl₂.6H₂O, Sigma Aldrich) for 24 h. Cells were then washed and incubated with AA for 24 h.

Human follicular fluids were collected as residual samples during the assisted reproductive techniques of oocyte collection from female ovaries by transvaginal ultrasound-guided aspiration using a single aspiration needle (Cook Medical, Bloomington, IN, USA) and phenotypically analysed to assess their mesenchymal properties (Figure S1) [15 (link)]. Mesenchymal stem cells present in the hFF were isolated by centrifuging the follicular fluid by density gradient separation (Lymphoprep, NycomedPharma, Oslo, Norway) for 30 min at 1800 rpm (to eliminate red blood cells and debris), as reported in [15 (link)]. hFF-MSCs were cultured at 37 °C in a humidified incubator with 5% CO₂ in DMEM medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS, Eurobio, Les Ulis, France), 2 mM of L-glutamine (Eurobio), 100 mg/mL of penicillin (Eurobio) and 100 μg/mL of streptomycin (Eurobio). Cells were used for all these experiments at the first passage of cell culture.

The human leukemic cell line K562 (from a patient with chronic myeloid leukemia (CML)) was obtained from American type culture collection (Manassas, VA, USA). The human leukemic cells lines U937 (from a patient with monocytic differentiation-inducible histiocytic lymphoma) and Jurkat (from the peripheral blood of a 14-year-old boy with T-cell acute lymphoid leukemia) were provided by Dr. Hélène Moins‐Teisserenc (Laboratoire d'Hématologie Biologique, AP‐HP, Hôpital Saint Louis, 75010 Paris, France). Cells were grown in RPMI-1640 (Eurobio, Toulouse, France) supplemented with heat-inactivated fetal bovine serum (10%; Eurobio), 2 mM L-glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin, 0.01 M Buffer Hepes, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO₂. Cell lines were proven mycoplasma-free using a MycoProbe Mycoplasma Detection Kit (R&D Systems) and maintained with ciprofloxacin (0.5 μg/mL).

A polydimethylsiloxane (PDMS) micro-grid array (Microsurfaces, Australia) of 4096 micro-wells (50 μm-sided) was placed in a MatTek dish (MatTek Corporation, Slovak Republic). It was covered by complete Roswell Park Memorial Institute (RPMI) 1640 Medium Glutamax plus (RPMI; Gibco-Invitrogen, France) supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Germany), 100 μg/mL penicillin, and 100 μg/mL streptomycin (both from Eurobio, France), 50 μM 2-mercaptoethanol (Gibco-Invitrogen), and 30 U/mL IL-2 (Prometeus Laboratories) and 20 ng/mL TGF-β (Miltenyi Biotec SAS, France). The suspension of activating beads (see above) was added at a concentration that allowed the highest possible number of micro-wells with a single bead. Then, the suspension of 1.5 × 10⁴ cells was added, again at a concentration to get high number of wells with a single cell. As a result of this procedure, a large number of micro-wells with a single bead and a single cell were obtained together with micro-wells with cells without beads or two or more beads. We define a clonal population as being the cells in a micro-well that had one cell at the beginning of the experiment. The cells were maintained at 37 °C, 5% CO₂.

Sp2/0-Ag14 cells (mouse B cell myeloma) were obtained from American Type Culture Collection^™ (ATCC-CRL-1581). L929 cells (L cell, L-929, a derivative of strain L, ATCC1 CCL-1) were kindly donated by C. Parra of the Facultad de Medicina at Universidad Nacional de Colombia. Peripheral blood mononuclear cells (PBMC) were isolated from a human donor using Ficoll-Paque^™.
The present study had prior approval of the Ethics Committee of the Facultad de Medicina at Universidad Nacional de Colombia.
All cell lines were cultured using Corning cell culture flasks (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco´s Modified Eagle Medium (DMEM) or RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Eurobio, Les Ulis, France) and 100 μg/ml streptomycin and penicillin (Eurobio, Les Ulis, France).
All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂. The culture medium was changed every 3 days. To assess population doubling time, the culture medium was discarded and the cells washed 3 times with PBS containing 0.03% EDTA before incubation with PBS-EDTA containing trypsin (1 µg/ml) for 5 min at 37 °C. The detached cells were immediately dispersed in culture medium supplemented with FBS and subcultured in a new flask for estimating PDT.

The human leukemia cell lines Nalm-6 and Reh (DSMZ®, Deutschland) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM of L-glutamine with 5000 UI/L penicillin and 50 mg/L streptomycin (Eurobio®, France), and maintained at 37°C in a 5% CO₂ humidified atmosphere. Fura-2/AM, Rhod-2/AM, Bapta-AM, U73122, SKF96365 and thapsigargin were purchased from Abcam Biochemicals, France. Poly-D-lysine, dimethylsulfoxide (DMSO), 2APB and dexamethasone were purchased from Sigma Aldrich, France. PD98059 was purchased from Euromedex, France. Antibodies to total (Erk1/2) and phosphorylated-Erk1/2 (pErk1/2) were obtained from Cell signaling Inc, France.