3h thymidine

To measure proliferation of cell lines, 3 × 10⁴ cells/0.2 mL medium were seeded into flat-bottom 96-well plates with indicated concentrations of diclofenac, diflunisal, metformin and cytarabine. To analyze immediate anti-proliferative effects, 0.5 μCi/0.2 mL ³H-thymidine (Amersham Pharmacia, Piscataway, NJ, United States) was added after 2 h and ³H-thymidine incorporation was determined after 24 h. In a second set of experiments, cells were labeled after 24 h and cultured for another after 24 h (48 h total).

PBMCs were isolated from heparinized blood by density sedimentation (Histopaque-1077; Sigma-Aldrich) as described previously (28 (link)). The PBMCs (1 × 10⁶ cells/200 μl/well) were cultured in triplicate in a 96-well flat-bottom plate (Nunc) and stimulated with SLA (10 µg/ml) or EF1-α (2.5 µg/ml) for 72 h at 37°C at 5% CO₂. After 72 h, supernatants were analyzed for IFN-γ and IL-12 by ELISA (BD Pharmingen), according to the manufacturer’s instructions. The remaining cells were pulsed with 1 μCi of [³H]-Thymidine (Amersham Biosciences) per well and incubated for another 16–18 h and harvested on glass fiber paper. Thymidine incorporation was measured after incubation for 16–18 h in a β-scintillation counter (Beckman Instruments) (24 (link)).

Total or depleted PBMCs were cultured in 96-well plates at a concentration of 1 × 10⁶ cells/mL in a final volume of 200 μL of complete medium containing RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine (Sigma, St. Louis, MO), 100 U/mL penicillin (Sigma, St. Louis, MO), 100 μg/mL streptomycin (Sigma, St. Louis, MO), and 10% (v/v) heat-inactivated human AB serum (Sigma, St. Louis, MO), 1% HEPES (0.01 M), (Invitrogen, Cergy-Pontoise, France), 1% sodium pyruvate (1 mM) (Invitrogen, Cergy-Pontoise, France), 1% MEM Non-Essential Amino Acids (Invitrogen, Cergy-Pontoise, France), 1‰ 2-Mercaptoethanol (10⁻²M), (Invitrogen, Cergy-Pontoise, France), and 0.2% gentamicin (20 μg/mL) (Invitrogen, Cergy-Pontoise, France). PBMCs were stimulated with LmES (10 μg/mL) or SLA (10 μg/mL) for five days. The uptake of [³H]-thymidine (Amersham, Saclay, France) was measured after adding 1 mCi/well for the last 6 h and evaluated for cell proliferation in a liquid scintillation counter (Rack Beta, LKB Wallace, Australia). Results were expressed as a proliferation index: mean counts of triplicates in antigen-stimulated cultures/mean counts of triplicates in unstimulated cultures.

The effect on primary human renal mesangial cell proliferation of DS-EA was assessed by [3H]-thymidine incorporation. Quiescent cells were treated with 10 μM of angiotensin II and DS-EA, and then 1 μCi of [3H]-thymidine (methyl-[3H] thymidine, 50 Ci/mmol; Amersham, Oakville, ON, Canada) was added for 24 h. Cells were extracted three times with cold 10% trichloroacetic acid (TCA) for 5 min and solubilized for at least 30 min in 0.3 N of NaOH. After harvesting, [3H]-thymidine activity levels were measured using a liquid scintillation counter (Beckman LS 7500, Fullerton, CA, USA). Each experiment was performed in triplicate or quadruplicate.

Purified CD4⁺CD25^– cells (responder cells) were either directly plated or preloaded with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then seeded into 96-well round-bottomed plates (Corning Costar Corp, NY) in triplicate at a density of 5 × 10⁴ cells/well. Cell proliferation was induced by stimulation with anti-CD3 (1 μg/ml) in the presence of irradiated allogeneic PBMCs as APC (2×10⁵/well, irradiated at 35 Gy) for 4 days. Proliferation was measured either by monitoring CFSE dilution or by thymidine incorporation, in which ³H thymidine (0.5 μCi/well, Amersham, Freiburg, Germany) was added to the culture 10 hours prior to cell harvesting.
To observe the inhibitory effect on cell proliferation, CD25⁺CD3⁺CD56⁺ cells directly purified from TILs, FOXP3-transduced CD3⁺CD56⁺ cells, TGF-β1-treated CD3⁺CD56⁺ cells, or CD4⁺CD25^high conventional Treg cells purified from PBMC were added to the assays at different ratios to responder cells from the same donor. Relative suppression was calculated as: % suppression = [1 – % of proliferation in coculture/% of proliferation in responder cells culture alone] × 100%. In some settings, the cells to be tested were separated from responder cells using transwell (0.4 μm, Corning Costar Corp). In other settings, exogenous rhIL-2 was added to the culture at a concentration of 10 ng/ml.

The [3H]thymidine (Amersham Biosciences, UK) incorporation was performed as previously described33 (link). Briefly, COLO-205 were applied to 24-well plates in growth medium (RPMI-1640 supplemented with 10% FBS, 100 ng/mL kanamycine). After COLO-205 had grown to 60–70% confluence, they were rendered quiescent by incubation for 24 h in RPMI-1640 containing 0.04% FBS. RPMI-1640 supplemented with 10% FBS and PBS with or without FA (0.1–10 μM) was added to the cell and the culture was allowed to incubate for 24 h. During the last 3 h of the incubation, [3H]thymidine was added at 1 μCi mL⁻¹ (1 μCi = 37 kBq). Incorporated [3H]thymidine was extracted in 0.2 N NaOH and measured in a liquid scintillation counter.