Fcs express v4

Monocytes were differentiated towards DC, M1‐ and M2 macrophage‐like cells in the absence or presence of hAMSC (as described above). After culturing, the monocytes were collected by gently scraping cells from the wells. Surface phenotype was investigated by flow cytometry as previously reported 33, using cell markers described to be expressed by DC 43, 44, M1‐ 45 and M2 45, 46 macrophages. Cells were acquired with a FACSAria (BD Biosciences) and analysed with the FCS express v4.07 (DeNovo Software). Antibodies and suppliers used in this study are described in Table 2.

Co‐cultures of T cells with N‐ and PE‐hAMSC were established in direct cell‐to‐cell contact. Around 10⁵ hAMSC were seeded in 96‐well plates (Nunc, Roskilde, Denmark) in 150 μl of UltraCulture medium (Lonza, Basel, CH, Switzerland) and irradiated (30 Gy) to block proliferation. The day after, mixed lymphocyte cultures (MLC) were obtained by culturing 10⁵ T lymphocytes and 10⁵ γ‐irradiated allogeneic PBMC in 100 μl of UltraCulture medium (Lonza), in the absence (controls) or presence of hAMSC. T‐cell proliferation was assessed by 5‐ethynyl‐2′deoxyuridine (EdU) incorporation as previously described 38. Briefly, 10 μM EdU (Life Technologies, Carlsbad, CA, USA) was added on day 5 and incubated for 16–18 hrs. Incorporated EdU was detected by the Cu‐catalysed alkyne‐azide cycloaddition (CuAAC or ‘click') reaction of the ethynyl group with 2.5 μM 3‐azido‐7‐hydroxycoumarin (Jena Biosciences, Jena, Germany), in buffer solution (100 mM Tris‐HCl pH 8.0, 10 mM L‐ascorbic acid, 2 mM CuSO₄) at RT for 30 min. The samples were acquired with a FACSAria (BD Biosciences) and analysed with FCS express v4.07 (DeNovo Software).

To evaluate the validity of some of the selectively expressed markers, cell surface expression levels of CD206/MRC1 and CD207/Langerin were assessed using flow cytometry through a similar gating strategy to the one presented in Fig. 1. Fluorescence minus one (FMO) controls were applied and net percentage positive cells was calculated by subtracting positivity for the FMO control. Results were analyzed using FCS Express v4.0 (De Novo Software, Los Angeles, CA).

The testes from rats treated with different concentrations of BPA for 24 h were removed, decapsulated and placed in PBS containing 0.1 mg/ml collagenase (Sigma, St Louis, MO). Then, the tubules were washed three times in PBS. Tubule cell isolation was performed by mechanical disaggregation in the presence of 0.1 mg/ml DNase (Sigma, St Louis, MO) using a 21G needle from different segments of the seminiferous tubules that were previously isolated in PBS. Then, the solutions were filtered by through a mesh with a pore diameter of 200 µm and another with a pore diameter of 70 µm. To analyze cell cycles, the individual cells were fixed in 70% ethanol overnight. The cells were pelleted and washed once with PBS. Then, the pellet was then dissolved in a cell cycle buffer containing 0.1% sodium citrate, 0.3% Triton X-100 (Sigma, St. Louis, MO, USA), 50 mg/ml propidium iodide and 50 mg/ml RNase A (Invitrogen, Carlsbad, CA, USA) dissolved in distilled water. The samples were analyzed within 10 min of buffer addition on a flow cytometer (FAScanto) and 10,000 gated events were acquired for each sample. All data were analyzed with the software FCS express V4.0 (De Novo Software, Los Angeles).

LNCaP cells treated with 100 µM BPA or 50 µM NP for 6 h were washed twice with PBS. After that, they were incubated with 5 µL of FITC-Annexin-V and 5 µL of propidium iodide for 15 min at room temperature in the dark. Then, 400 µL of binding buffer was added and analyzed by flow cytometry (FAScanto, BD Biosciences, Franklin Lakes, NJ, USA) and 10,000 gated events were acquired in each sample. All data were analyzed with software FCS express V4.0 (De Novo Software, Los Angeles).

Tubule cells isolation was performed by mechanical disaggregation in the presence of 0.1 mg/ml DNasa (Sigma, St Louis, MO) using a 21G needle from different segments of the seminiferous tubules isolation previously in PBS. Then, the solutions were filtered by through a mesh with a pore of 200 μm and then a pore of 70 μm of diameter. The filtered solutions were centrifuged for 3 min at 800 × g. Then the isolated cells were incubated in DMEM-F12 medium containing 3% BSA, for 30 min at 4°C. Primary antibody against ADAM17, or against ADAM10 coupled to phycoerytrin, was added diluted in blocking solution (1:250) and left to incubate overnight. The next day cells were washed three times with PBS and in the case of those incubated with the antibody against anti-ADAM17 they were dissolved in blocking solution with the corresponding secondary antibody conjugated with FITC (diluted 1:250) and incubated for one hour at 4°C. Then, cells were washed three times with PBS and the final pellet dissolved in PBS. The samples were analyzed by a flow cytometer (FAScanto) and 10,000 gated events were acquired in each sample. As controls, one autofluorescence sample, one sample with only primary antibody and one sample with only secondary antibody were analyzed. All date were analyzed with software FCS express V4.0 (De Novo Software, Los Angeles).

After treatment, LNCaP and A2780 cells were fixed in 70% ethanol. The cells were then pelleted and washed once with PBS. Then, the pellet was dissolved in a buffer containing 0.1% sodium citrate, 0.3% Triton X-100 (Sigma), 50 mg/mL propidium iodide, and 50 mg/mL RNase A (Invitrogen, Carlsbad, CA, USA). The samples were then analyzed by a flow cytometer (FAScanto, BD Biosciences, Franklin Lakes, NJ, USA). Ten thousand gated events were acquired in each sample. All data were analyzed with software FCS express V4.0 (De Novo Software, Los Angeles, CA, USA).