Cd34 alexa fluor 647 ram34

After obtaining SVF single cell suspensions, different cell populations were sorted as described previously (Bayindir et al, 2015). Cells were preincubated with FcBlock (anti‐CD16/32; eBioscience) for 10 min on ice. Erythrocytes were depleted via magnetic separation with an OctoMACS Separator according to the manufacturer's instructions, following incubation with Anti‐Ter119 MicroBeads (130‐049‐901, Miltenyi Biotec) for 15 min on ice. The flow‐through was stained with CD45‐FITC (30‐F11, eBioscience), CD31‐eFluor450^® (390, eBioscience), CD29‐PerCP‐eFluor^® 710 (HMb1‐1, eBioscience), CD34‐Alexa Fluor^® 647 (RAM34, BD Biosciences, Heidelberg, Germany), and Sca‐1‐Alexa Fluor^® 700 (D7, eBioscience) for 30 min on ice. Cells were washed, sorted as Lin(CD31/CD45)⁻CD29⁺CD34⁺Sca1⁺ with a BD FACS Aria (BD Biosciences), and centrifuged at 300 g for 5 min at 4°C and resuspended either in culture medium with bFGF for culturing or in QIAzol^® Lysis Reagent (Qiagen, Hilden, Germany) for RNA isolation. FCS files exported via BD FACSDiva™ software were analyzed with FlowJo Software (FlowJo, Ashland, OR).

Stromal-vascular fraction single cell suspensions in D-PBS (Life Technologies, Darmstadt, Germany) supplemented with 0.5% BSA and 1 mM EDTA (Sigma-Aldrich) were preincubated with FcBlock [anti-CD16/32 (93, eBioscience), Frankfurt, Germany] for 10 min on ice. Cells were then incubated with Anti-Ter119 MicroBeads (130-049-901, Miltenyi Biotec, Bergisch Gladbach, Germany) on ice for 15 min, to perform erythrocyte depletion by magnetic-activated cell sorting (MACS^®) with an OctoMACS Separator according to the manufacturer’s instructions. The flow-through was collected and stained with CD45-FITC (30-F11, eBioscience), CD31-eFluor 450 (390, eBioscience), CD29-PerCP-eFluor 710 (HMb1-1, eBioscience), CD34-Alexa Fluor 647 (RAM34, BD Biosciences, Heidelberg, Germany), Sca-1-Alexa Fluor 700 (D7, eBioscience), and CD140a(Pdgfrα)-biotin (APA5, eBioscience) for 30 min on ice, followed by staining with streptavidin-PE-Cy7 (eBioscience). After antibody staining, samples were washed and sorted with a BD FACS Aria (BD Biosciences). Unstained cells as well as FMO stainings were used as negative controls. Single-stained controls were used for compensation. Data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA). Sorted cells were centrifuged at 300 × g for 5 min for further processing.