Anti hla dr

For the analysis of MHC molecules expression by SARC-L1, cells were stained with the
following fluorescent conjugated monoclonal antibodies: Anti-2K^b, (clone
AF6-88.5.5.3,); anti-H-2K^dand anti-I^A/I^E(Ebiosciences, France); anti-HLA-A2 (BD Biosciences, France); anti-HLA-DR (Diaclone,
France) or with their respectively isotype control. For tumor infiltrating immune
cells analysis, tumor cells were mechanically dissociated (GentleMax, Miltenyi,
France) tumor infiltrating lymphocytes were separated on a Percoll (Sigma-Aldrich,
France) density gradient. Lymphocytes layer was collected and stained with the
following fluorescent conjugated monoclonal antibodies: anti-CD3, anti-CD4, anti-
CD8, anti-Gr1, anti-PD-L1 (Biolegend, France), anti-CD11b, anti- FoxP₃,
anti-CD25 (eBiosciences, France), anti-PD-1, and anti-TIM-3 (Miltenyi, France),
fixable viability stain (BD, France). The tumor cells fraction was stained
withanti-CD45, anti-PD-L1, anti-HLA-A2 and anti-HLA-DR (BD Biosciences, France) and
fixable viability stain (Biolegend, France). Samples were acquired on a FACS Canto II
(BD Biosciences, France) and analyzed with the BDFacsDIVA or FlowJo software.

The DPMSCs, UCMSCs, and ADMSCs were checked for their surface marker profile by FACSCalibur flow cytometry system (Becton Dickinson, CA, USA) as already described [8 (link), 9 (link), 23 (link)]. Briefly, the MSCs were detached from the surface of the flask with enzyme digestion for 3 minutes at room temperature, collected, and centrifuged at 300g for 5 minutes. The pellets were resuspended in stain buffer and the cells were counted by hemocytometer. Then, 2.5 × 10⁵ cells were incubated for 45 min, in the dark at 4°C, with the following antibodies: fluorescein isothiocyanate- (FITC-) labeled mouse antihuman CD90 (StemCell Technologies, Milan, Italy), CD105, CD14, and CD19 (Diaclone, France); R-phycoerythrin- (PE-) labeled mouse antihuman CD34, CD44, CD45 (Diaclone, France), and CD73 (Becton Dickinson, CA, USA); and anti-HLA-DR (Diaclone, France). The control for FITC- or PE-coupled antibodies was isotypic mouse IgG1. The data were evaluated using CellQuest software (Becton Dickinson, CA, USA).

The cells obtained were checked for their staminal profile by FACSCalibur flow cytometry system (Becton Dickinson, CA, USA). In agreement with minimal criteria for the identification of human MSCs (Dominici et al., 2006 (link)), 2.5 × 10⁵ cells were removed with Dulbecco Phosphate buffer saline (D-PBS) and were then stained for 45 min with the following antibodies: fluorescein isothiocyanate-(FITC)-labeled mouse anti-human CD90 (StemCell Technologies, Milan, Italy), CD105, CD14, CD19, (Diaclone, France), R-phycoerythrin-(PE)-labeled mouse anti human CD34, CD45 (Diaclone, France), CD73 (Becton Dickinson, CA, USA), and anti HLA-DR (Diaclone, France). The control for FITC- or PE-coupled antibodies was isotypic mouse IgG1. The data were evaluated using CellQuest software (Becton Dickinson, CA, USA).