Sorp facsaria2

10% of a FcR blocking reagent (Myltenyi Biotec) was added and incubated for 10- 30 minutes on ice. 100 μl of staining buffer was added per 10⁷ of cells. The PBMCs were then stained with the following antibodies: mouse anti-human IgG2b CD14 –APC and mouse anti-human IgG1 CD16-PE (BD bioscience) and incubated for 30–45 minutes on ice in the dark. Finally, the cells were spun at 200 × g for 15 minutes and the pelleted PBMCs were resuspended in 1 mL of staining buffer for sorting. Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences). Data were processed with FACSDiva 6.3 software (BD Biosciences). Doublets were excluded by FSC-W × FSC-H and SSC-W × SSC-H analysis (Supplementary Figure 4). PE fluorescent was acquired by 498 nm blue laser and 575/26 nm emission; APC fluorescence was obtained by 650 nm red laser and 660/20 nm emission; Figures display the median of fluorescence intensity (mfi) relative to control. Single stained channels were used for compensation and fluorescence minus one (FMO) controls were used for gating. Purity of the sorting was controlled after each sorting and was > 90%.

Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences) as previously described [13 (link), 15 (link)]. Data were processed with FACSDiva 6.3 software (BD Biosciences). Doublets were excluded by FSC-W x FSC-H and SSC-W x SSC-H analysis. eGFP fluorescence was acquired with 488 nm blue laser and 510/50 nm emission, EpCam APC conjugated (BD Biosciences) was acquired with 647 nm red laser and 670/14 nm emission, Pkh red fluorescence was acquired with 535 nm green laser and 582/15 nm emission. Charts display the median of fluorescence intensity (mfi) relative to control. Single stained channels were used for compensation and fluorophore minus one (FMO) controls were used for gating. 20,000 events were acquired per sample. Viability was assessed by flow cytometry evaluation of Calcein AM staining as described by the manufacturer (Live Dead Viability/Cytotoxicity Kit, Molecular Probes, Invitrogen).

Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences) as previously described [75 (link), 79 (link)]. Data were processed with FACSDiva 6.3 software (BD Biosciences). Doublets were excluded by FSC-W x FSC-H and SSC-W x SSC-H analysis. eGFP fluorescence were acquired with 488 nm blue laser and 510/50 nm emission, EpCam APC conjugated (BD Biosciences) was acquired with 647 nm red laser and 670/14 nm emission, WGA AF594 fluorescence was acquired with 535 nm green laser and 582/15 nm emission. Charts display the median of fluorescence intensity (mfi) relative to control. Single stained channels were used for compensation and fluorophore minus one (FMO) controls were used for gating. 20,000 events were acquired per sample.

Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences). Data were processed with FACS Diva 6.3 software (BD Biosciences) as previously described [19 (link)]. Doublets were excluded by FSC-W × FSC-H and SSC-W × SSC-H analysis; calcein-AM and Annexin V were acquired with 488 nm blue laser and 510/50 nm emission, PI was acquired 488 nm blue laser and 670/14 nm emission. Charts display the median of fluorescence intensity (mfi) relative to control. Single stained channels were used for compensation and fluorophore minus one (FMO) controls were used for gating. 20,000 events were acquired per sample.

Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences). Data were processed with FACS Diva 6.3 software (BD Biosciences) as previously described [39 (link), 40 (link)].

Fluorescence (FL) was quantified on a SORP FACSAria2 (BD Biosciences) as previously described [14 (link), 16 (link)]. Data were processed with FACSDiva 6.3 software (BD Biosciences). Doublets were excluded by FSC-W x FSC-H and SSC-W x SSC-H analysis. Charts display the median of fluorescence intensity (mfi) relative to control. Single stained channels were used for compensation and fluorophore minus one (FMO) controls were used for gating. 20,000 events were acquired per sample.
Cells from ascites fluids were stained with EpCam APC conjugated (BD Biosciences) and the fluorescence was acquired with 647 nm red laser and 670/14 nm emission.
MSC were defined as Lin⁻CD45⁻CD90⁺CD73⁺CD105⁺CD29⁺. The cell suspension was stained with mouse anti-human CD45 antibody (BD biosciences, #339192, clone 2D1) coupled with Amcyan, anti-mouse lineage cocktail 1 (Lin, BD biosciences, #340546, CD3, CD14, CD16, CD19, CD20, CD56) coupled with FITC, mouse anti-human CD105 (biolegend, #323212, clone 43A3) coupled with AF647, mouse anti-human CD73 (BD biosciences, #550257, clone AD2) coupled with PE, mouse anti-human CD29 (biolegend, #323212, clone TS2/16) coupled with APC-Cy7, mouse anti-human CD90 (BD biosciences, #550402, clone 5E10) coupled with AF700.

Cells were harvested and blocked in PBS-5% FBS-1% BSA-10% FcR Blocking Reagent (Myltenyi Biotec). Single-cell suspension was analyzed and sorted on SORP FACSAria2 (BD Biosciences). Data were processed with FACSDiva 6.3 (BD Biosciences). Doublets were excluded by FSC-W × FSC-H and SSC-W × SSC-H analysis, single stained channels were used for compensation, and fluorophore minus one (FMO) controls were used for gating. eGFP fluorescence was acquired with 488 nm blue laser and 510/50 nm emission, 50 000 events were acquired per sample. Charts display the median of fluorescence intensity (mfi) relative to control. During cell-sorting purity-phase mask was applied. OCC monocultures were processed and sorted as controls.