Lysis 2 software

Binding of monomeric IgG to B2KA cells expressing FcγRI was measured as previously described 1 (link) except that, for the B2 Abs, 80 μg/mL biotin-conjugated goat anti-human λ-chain Abs (Sigma, Poole, UK) were used as the first detection reagent.
Binding to FcγRII and III receptors was measured by pre-complexing the Abs with equimolar amounts of F(ab’)₂ fragments, which recognised the light chain 4 (link): goat F(ab’)₂ anti-human κ (Rockland) for Fog-1 and goat anti-human λ-chain F(ab’)₂ molecules (AbD Serotec or Rockland) for B2 Abs. Human IgA1,κ purified myeloma protein (The Binding Site, Birmingham, UK) or IgA,λ (Jackson ImmunoResearch, Newmarket, UK) were used as negative control test Abs. Complexes were detected using FITC-conjugated F(ab’)₂ fragments of rabbit anti-goat IgG, F(ab’)₂-specific Abs (Jackson ImmunoResearch) or FITC-conjugated donkey anti-goat IgG Abs (AbD Serotec).
Levels of fluorescence were determined using a CyAn ADP flow cytometer and Summit v4.3 software (DakoCytomation, Ely, UK) or on a FACScan flow cytometer and LysisII software (Becton Dickinson, Oxford, UK).

RASF were plated into six-well plates at 1 × 10⁵ cells per well in complete media. Cells were incubated at 37 °C for 48 h. RASF were incubated with 0.3 μg/mL of phycoerythrin-conjugated mouse monoclonal anti-intracellular adhesion molecule (ICAM)-1 or isotype-matched IgG control (all Becton Dickinson, Franklin Lakes, NJ, USA) for 30 min at 4 °C. Cells were then washed twice and fixed in 1% paraformaldehyde, and analysed by flow cytometry (FACS) using a FACScan flow cytometer and Lysis II software (both from Becton Dickinson). Adhesion marker expression was calculated based on the median fluorescence intensity (MFI), with background MFI (unstained samples) subtracted. Technical replicates were averaged to determine the MFI per RASF donor.

Apoptosis was assessed using an Annexin V-FITC/PI Apoptosis Detection Kit. For FCM analysis, 2×10⁵ cells/well were treated with different concentrations of RGZ (0, 20, 40, 80 µg/ml) or GW9662 (0, 5, 10, 20 µg/ml). The cells were subsequently collected, pelleted and washed with phosphate-buffered saline (PBS) prior to fixing overnight at −20°C in 75% ethanol. The cells were washed again with PBS, resuspended and treated with RNase (200 mg/l) for 30 min at 37°C, prior to incubation with 20 mg/l PI in the dark for 15 min. The suspension was passed through a nylon mesh filter and underwent FCM analysis (FACSort^™; Becton-Dickinson, Franklin Lakes, NJ, USA). All data were collected, stored and analyzed by LYSIS II software (Becton-Dickinson). The experiments were repeated three times, and the results are presented as the mean ± standard deviation (SD).

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).