Facsaria 2 cytometer

Monocytes were co-cultured with VIP siRNA transfected Swan 71 cells or Srcbl siRNA transfected cells as control, and after 20 hours cells were recovered by TrypLe^TM treatment (Life technologies, Grand Island, NY, USA) and stained with FITC, PE-Cy5, APC or PE-conjugated mAbs directed to surfer markers (CD14, CD86, CD39, CD16) or intracellular cytokines (IL-10, IL-12) (BD Pharmingen, San Diego, CA, USA). For intracellular cytokine detection Stop Golgi was added to the medium in the last 4 h of co-culture following manufacturer’s instructions to promote intracellular accumulation. Then cells were recovered and, after washing with staining buffer, they were stained with mAb anti-superficial molecules, washed, fixed and permeabilised with the Fix/Perm kit as manufacturer recommended. Permeabilised cells were incubated for 30 min with IL-10 or IL-12. Cells were finally washed with PBS-2% FBS. Ten thousand events were acquired in a FACS Aria II cytometer^® (Becton Dickinson, San José, CA, USA) results were analyzed using FlowJo software (http://www.flowjo.com/) expressed as MFI or double positive cell frequencies by flow cytometry. For monocyte profile, positive cells were determined inside the electronically gated CD14 positive cell population previously selected in FSC vs. SSC as previously39 (link).

Bone marrow cells were collected from femurs and tibiae. Staining of PE‐conjugated anti‐CD99 (BioLegend), APC‐conjugated anti‐CD45 (BioLegend), and APC‐conjugated Ter119 (BioLegend) was carried out at room temperature for 20 minutes. Cells were then washed in phosphate‐buffered saline at 1200 rpm for 5 minutes and resuspended in 400 µL of phosphate‐buffered saline. Samples were analyzed on a FACS Aria II cytometer (Becton Dickinson) or JSAN cell sorter (Bay Bioscience), and data were analyzed by FlowJo 8.7 software (Tree Star).

Trophoblast cell lines were incubated with Stop Golgi (Becton Dickinson, San José, CA) for the last 4 h of cell culture following manufacturer’s instructions to promote intracellular accumulation of proteins. After washing with PBS-2% FBS (staining buffer), cells were fixed and permeabilized with the Fix/Perm kit according to manufacturer’s instructions (Becton Dickinson, San José, CA). Permeabilized cells were incubated for 30 min with human VIP monoclonal antibody at room temperature. Cells were washed with staining buffer twice and then stained with a secondary antibody Alexa 488 for 45 min. Ten thousand events were acquired in a FACS Aria II cytometer^® (Becton Dickinson, San José, CA, USA) and the data was analyzed using the FlowJo software (http://www.flowjo.com/). Results were expressed as fold increase of VIP positive cells respect to the isotype control.

EPI is a single molecule capable of emitting fluorescence detectable by flow cytometry (550–600 nm). This property allows the determination of EPI intracellular accumulation as we previously described [29 (link)]. MDA-MB-231 cells (5 × 10⁵) were transfected and treated as mentioned above and EPI fluorescence was collected through a 564–606 nm band-pass filter. Samples were analyzed using a FACS Aria II cytometer and data was evaluated using FlowJo 5 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

SHED and MSC-like cells from iPS-SHED and from iPS-FIB were harvested and resuspended to 10⁵ cells in 100 uL of PBS containing 1% BSA. Cells were separately labeled with FITC, PE, PE-Cy5, PERCP-Cy5.5, or APC-H7 conjugated rat anti-human antibodies CD29, CD31 (Biolegend), CD34, CD45, CD73, CD90 CD105, and CD166 (Becton Dickinson) on ice and protected from light for 40 min. An isotype-matched mAb was used as a control (Becton Dickinson). Data were acquired and analyzed with the FACSAria II cytometer and CellQuest software (Becton Dickinson). Multipotential differentiations of MSC-like cells from iPS-SHED and from iPS-FIB were performed as previously described by de Mendonça Costa et al., 2008 [13 (link)], and representative pictures of adipogenesis, osteogenesis, and chondrogenesis were included as supplementary Figure  2.

4 × 10⁴ Swan 71 or BeWo cells were seeded until subconfluence. 50 nM VIP/100 µM rapamycin were added for 6 h or 24 h respectively in DMEM-F12 2% FBS. For VIP detection, protein secretion was inhibited by Stop Golgi incubation for 4 h. Cells were trypsinized, fixed and permeabilized with the Cytofix/Cytoperm^TM plus kit according to manufacturer’s instructions (Becton Dickinson, San José, CA, US). Cells were washed twice and then incubated for 1 h with mouse anti SNAT1 or rabbit anti VIP (Abcam, Cambridge, UK), mouse anti GLUT3 (Santa Cruz Biotechnology, Dallas, TX, US) or rabbit anti mTOR (Cell Signaling, Massachusetts, US) monoclonal antibodies at room temperature. Cells were washed twice and then incubated with secondary antibody anti mouse-Alexa 488/anti rabbit-Alexa 647 (ThermoFisher Scientific, Massachusetts, US) for 40 min at room temperature. After washing twice with permwash buffer and resuspended in FACS solution, ten thousand events were acquired in a FACS Aria II cytometer® (Becton Dickinson, San José, CA, US) and the data was analysed using the FlowJo software (http://www.flowjo.com/).