Cd271 apc

SVF cells, from paired samples of LT and SAT of MSL patients, were freshly isolated for an ex vivo multiparametric flow cytometry analysis to quantify and characterize the ASCs present in the SVF, avoiding modifications due to plastic adhesion and culture conditions. Cells (1 × 10⁵) were washed with cold FACS buffer (2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)), collected by centrifugation (350× g, 10 min) and simultaneously incubated in the dark for 10 min at room temperature with the following monoclonal mouse anti-human fluorochrome-conjugated antibodies against the indicated surface markers in different combinations, as reported in Table 3: CD31-FITC and CD31-PE, CD45-FITC, CD34-PerCP-Cy5.5, CD73-APC, CD90-PE, CD271-APC and CD146-PE (BD Biosciences, San Jose, CA, USA). Labeled cells were acquired and analyzed by a FACSCanto^TM Flow Cytometer (BD Biosciences) using the BD FACSDiva™ software, as detailed previously by Belligoli et al. [45 (link)], to quantify and characterize the mature endothelial cells (CD45−CD31+CD34−), endothelial progenitor cells (CD45−CD31+CD34+) and ASCs (CD45−CD31−CD34+) [46 (link),47 (link)].

Flow cytometry analysis of MSC surface markers was performed as described [42 (link)]. Briefly, cells were stained with monoclonal antibodies, anti-human CD73-PE, CD-34-APC, CD146-PEcy7, CD271-APC, Streptavidin-PEcy7, CD140a-PE (BD Pharmingen), SSEA4-Biotin (R&D Systems, Minneapolis, MN), CD166-FITC (Serotec, Oxford, UK) and analyzed using FACSCalibur (Becton Dickinson) and CellQuest software. To examine the expression of cross-talk molecules, MSCs were permeabilized and intracellular stained with specific antibodies against Jagged-1(28H8, Cell signaling, Danvers, MA) or CXCL-12 (79018, R&D Systems) as described [13 (link), 43 ]. Relative expression levels were determined by ΔMFI, difference in mena fluorescent intensity. Osteogenic differentiation and adipogenic differentiation of MSCs were induced by each specific differentiation medium and quantified by Alizarin Red staining or lipid droplet as previously described [19 (link)]. For colony formation (CFU-F), MSCs were plated at a density of 500 cells per 100 mm dish, and after incubation for 14 days, the number of colonies containing >50 cells was counted after staining with crystal violet (Sigma) in methanol.

At passage 3–4, 3 × 10⁵ cells were washed with PBS containing 0,1% sodium azide and 3% FBS and then incubated for 30 min in ice and in the dark with the following mouse anti-human monoclonal antibodies directly conjugated with fluorochromes: CD13/FITC (22A5, Caltag Medsystems, UK), CD14/PE (M5E2, BD Biosciences, CA, USA), CD34/FITC (581, BD Biosciences), CD45/ALexa405 (HI30, Caltag Medsystems), CD90/PECy5 (5E10, BioLegend, CA, USA), CD105/APC (45A5A3, BioLegend), CD146/PE (P1H12, BD Biosciences), CD271/APC (C401457, BD Biosciences). Unstained cells were used as controls. Data was acquired using FACSCanto II (BD Bioscienses) flow cytometer and at least 20.000 events were analyzed with FACSDiva (BD Biosciences) or FlowJo 7.6.5 (FlowJo.com; Tree Star, OR, USA) software.