Anti ter119 microbeads

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).

For cell isolation and sorting, BM cells from both hind limbs were collected and single cell suspension was prepared. Ter119⁺ BM cells were isolated using anti-Ter119 microbeads (Miltenyi Biotec) following manufacturer’s protocol. For cell sorting, BM cells were incubated with anti-CD71 and anti-Ter119 antibodies. CD71⁺Ter119^-, CD71⁺Ter119⁺ and CD71^-Ter119⁺ erythroid cells were sorted by using a FACSAria (BD Biosciences) flow cytometer.

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.

Bone marrow cells were stained with purified anti-Gr-1, CD3, TER-119, B220, CD19 and CD11b mAbs and labeled cells were depleted with anti-rat IgG microbeads using LD columns with Quadro MACS magnet (Miltenyi Biotec, Gladbach, Germany). FL cells were depleted of erythroid cells using anti-TER-119 microbeads (Miltenyi Biotec). For detection of CLPs and KSL cells, cells were stained with FITC-lineage markers as well as PE-anti-Sca-1, AlexaFluor 647-anti-IL-7Rα, and PE-Cy7-anti-CD117/c-kit mAbs. The FITC-lineage marker cocktail consisted of following Abs; anti-IgM (Southern Biotech, Birmingham, AL), B220, CD19, Gr-1, DX5, TER-119, CD3e. Stainings with biotinylated antibodies were developed by streptavidin-PE. Stained cells were analyzed on LSR II flow cytometer (BD Bioscience), or purified on FACS Aria cell sorter (BD Bioscience). Flow cytometry data were analyzed with FlowJo software (Tree Star, Ashland, OR).