Phytohaemagglutinin

Aggregation chimeric mosaics containing reversine-treated and control blastomeres were created at the eight-cell stage. Individual blastomeres were obtained as described above and carefully aggregated together in M2 containing 0.1 mg ml⁻¹ phytohaemagglutinin (Sigma). For live imaging from the eight-cell stage, the blastomeres were labelled just before chimera aggregation by incubation for 10 min in M2 containing a 1:100 dilution of FM4-64 (Invitrogen). For chimeras imaged from the mid-blastocyst stage, half of the embryos were labelled at the two-cell stage by microinjection of Tomato fluorescent protein mRNA into both blastomeres.

Detection of proliferation activity of whole blood and lymphoid organ cell suspensions was based on incorporation of ³H-thymidine as described previously [7 ]. For the lymphocyte proliferation assay, the density of the cell suspension was adjusted to 1 × 10⁶/mL in RPMI 1640 medium supplemented with 10% precolostral calf serum, 100 000 U/L penicillin and 0.2 g/L streptomycin. After 5 days of in vitro lymphocyte stimulation (37 °C, 5% CO₂) with live E. cuniculi spores (4 × 10⁵/well) or non-specific mitogens phytohaemagglutinin (PHA, 100 μg/mL), concanavalin A (ConA, 10 μg/mL) and pokeweed mitogen (PWM, all mitogens purchased from Sigma-Aldrich, St. Louis, MO, USA, 10 μg/mL) or without stimulant, 50 μL of medium with ³H-thymidine (5 μCi/mL) was added for the last 20 h. The incorporation of ³H-thymidine was analyzed with a microplate scintillation and luminescence counter (TopCount NXT™, Packard Bioscience Company, Meriden, CT, USA). The results were expressed in terms of stimulation indices (SI), which were calculated as the ratio of counts per minute (CPM) in stimulated samples versus CPM in non-stimulated ones.

Poly-L-lysine (PLL, MW = 1.5~3.0 k), mycophenolic acid (MPA), Cyclosporine A (Cyc A), rapamycin (RAPA), and phytohaemagglutinin (PHA) were purchased from Sigma-Aldrich. Dried Hyaluronic acid sodium (HA, MW = 10 k) was purchased from Lifecore Biomedical. Dimethyl sulfoxide (DMSO, 99.5%) was purchased from Daejung. The concentration of PLL (pH 8) and HA (pH 6) was 1 mg/mL in the co-solvent (DMSO: DI water 1:1 v/v). The concentration of MPA and Cyc A solutions were 0.1 mg/mL. The concentration of the RAPA solution was 20 ng/mL. PHA was dissolved in DMSO at 1 mg/mL. In general, DMSO plays an important role in cell culture, including use as a cryoprotectant for the frozen preservation of cultured cells, cell medium supplement to reduce ice formation, and a component of the polymerase chain reaction mix before reacting^{38 (link)–40 (link)}.

For isolation of CD4⁺ T lymphoblast cultures, human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors provided by “Centro de Transfusiones de la Comunidad de Madrid” under an agreement with the Hospital Princesa (Madrid) and approved by the CEIm of Hospital Princesa, according to government ethical consent. Upon separation on a Biocoll gradient (Biochrom, L6115), nonadherent cells were collected after plating PBMCs at 37°C and purified using Stem Cell Technologies EasySep kit for CD4⁺ T cells. Cells were then cultured for 48 h in the presence of SEE (0.01 μg/ml; Toxin Technology) and PHA (0.2 μg/ml phytohaemagglutinin, Sigma Aldrich) to induce lymphocyte proliferation, and IL-2 (50 U/ml) was added to the culture medium every 2 days. HuCD4⁺ T lymphoblasts were transfected 7 days after isolation. Experiments were performed 48 h post-transfection.
Jurkat E6-1 cell line (Vαl.2 Vβ8⁺ TCR) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Dr. A. Weiss. Jurkat E6-1 and Raji lymphoblastoid B cell line were grown in RPMI 1640 medium (Gibco-invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). Cells were cultured at 37 °C, in 5% CO₂ atmosphere. Jurkat E6-1 T cell clones were cultivated with 0.5 mg/mL^-1 G418.

Peripheral blood mononuclear cells (PBMCs) were either isolated from fresh blood by Ficoll-Hypaque gradient centrifugation or used after thawing. PBMCs (20×10⁶ cells) were cultured in RPMI medium containing 10% FBS and 2% gentamycin. Cells were stimulated with 1 ug/ml of phytohaemagglutinin (PHA; Sigma-Aldrich, St. Louis, Mo, USA) for 72 hours, washed in PBS (1×) and pelleted for RNA extraction. Total RNA (enriched for small RNA) was isolated according to manufacturer's instructions using the mirVana miRNA isolation Kit (Ambion, Huntingdon, UK). RNA concentration was calculated using NanoDrop technology ND-1000 (Thermo Scientific, Waltham, MA, USA). RNA integrity was then evaluated using RNA 6000 Nano LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All chips were prepared according to the manufacturer's instructions at the Genomic platform of the CCiTUB (Centres Científics i Tecnològics University of Barcelona) located at the Barcelona Science Park (PCB). Total RNA degradation was evaluated by reviewing the electropherograms and the RNA integrity number (RIN) of each sample. Only samples with preserved 18S and 28S peaks and RIN values greater than 7 were selected for miRNA profile analysis.