Staining buffer set

AFSCs at first passage were sub-cultured until reaching 80% confluence. As previously reported [51 (link)], following trypsin dissociation, cells were stained with the following antibodies (mAbs): Rabbit-Thy-1 (CD90) and Rabbit-Endoglin (CD105) (Millipore, CA, USA), Mouse-SSEA4 (Cell Signaling Technology, MA, USA), Goat-Integrin β1 (CD29), Rabbit-HCAM (CD44) (Santa Cruz Biotechnology, CA, USA), Mouse-5’-Nucleotidase (CD73) (Gene Tex, CA, USA).
The expression of surface markers was analysed by indirect staining using secondary fluorochrome Alexa 488-conjugated antibodies (Abcam, Cambridge, UK). Non-specific fluorescence was assessed by using the secondary antibody alone. A minimum of 5,000 cells per sample was acquired and analysed using FACScan flow cytometer and Lysis II software (both from Becton Dickinson, San Jose, CA, USA).
The same cell samples were analyzed for nuclear stem cell markers, such as Goat- Sox2 (Santa Cruz Biotechnology, CA, USA) and Rabbit-Nanog and Rabbiti-Oct4, (Cell Signaling, MA, USA), after Fixation/Permeabilization process performed with the Staining Buffer Set (Miltenyi Biotec Inc, Auburn, CA, USA) optimized for nuclear staining.
After exposure to adipogenic differentiation medium, nuclear differentiation marker rabbit anti-PPAR (Santa Cruz, CA, USA) was analysed by using the Staining Buffer Set (Miltenyi Biotec Inc, Auburn, CA, USA).

Tumor samples were mechanically dissociated and dissolved in staining buffer (PBS, 0.2% BSA, 0.02% NaN₃). For analysis of peripheral blood, red blood cells were lysed in lysing buffer (BD Biosciences). FcR blocking reagent (Miltenyi Biotec) was added, and each sample was incubated with antibodies in staining buffer rinsing solution plus 0.5% BSA (Miltenyi Biotec) according to the supplier’s instructions (Additional file 1). For intracellular labeling, a staining buffer set (Miltenyi Biotec) was used. After washing, cells were resuspended in PKH26 reference microbead solution (Sigma-Aldrich) and analyzed using multicolor flow cytometry (CyFlow space, Sysmex; LSR II or Fortessa X20, both BD Biosciences). Quantitative expression data of selected markers are presented as geometric mean fluorescence intensity (gMFI), cell type frequencies as the percentage of viable singlet cells of a defined population.