Cd14 fitc

Characterization of the SVF cell subpopulations was performed according to the recommendations of the International Federation of Adipose Therapeutics and Sciences (IFATS) and the International Society for Cellular Therapy (ISCT). Briefly, SVF suspension was digested with DNase I 10U/mL (Roche Diagnostics, Indianapolis, Indiana, USA) in Dulbecco’s Phosphate Buffered Serum (DPBS) Ca⁺⁺/Mg⁺⁺ free medium containing 0.1 mM ethylenediaminetetraacetic acid, 25 mM Hepes, 1% fetal calf serum for 15 minutes at 37°C and filtered through a 70-μm nylon cell strainer to eliminate the majority of cell aggregates. Cells were centrifuged, resuspended, and labeled 20 minutes at 4°C with the following fluorochrome-conjugated antibodies: CD14-FITC, CD90-FITC, CD146-PE, CD34-ECD, CD45-PC5 (Beckman Coulter, Miami, FL, USA) or their isotype control to determine nonspecific fluorescence. Red blood cells were lysed in NH₄Cl for 10 minutes at 4°C before cells were centrifuged and resuspended in DPBS Ca⁺⁺/Mg⁺⁺. NucBlue (Thermo Fisher Scientific, Waltham, MA, USA), allowing discrimination of viable and dead cells, was added for 5 minutes before flow cytometry analysis on a NAVIOS flow cytometer (Beckman Coulter). CD45 negative cells were discriminated in CD34⁻CD146⁺ and CD34⁺CD146⁻CD90⁺ described as regenerative perivascular cells, and CD34⁺CD146⁺ as endothelial cells.

Multiparameter flow cytometry of immune cells in PB and CSF samples was done as described previously (24 (link), 29 (link)). During lumbar puncture CSF was sampled into polypropylene tubes. All CSF samples were processed in < 20 min. Cells were isolated from CSF by centrifugation (15 min, 290 g, 4°C) and subsequent incubation in VersaLyse buffer (Beckman Coulter, Germany). PB samples were collected in EDTA monovettes and cells were isolated by using VersaLyse buffer. For immunostainings, the following fluorochrome-conjugated antibodies were used: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APC-Alexafluor700, CD16-APC-Alexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all from Beckman-Coulter). Data acquisition was performed with a Navios flow cytometer (Beckman-Coulter). Gating strategy for Leukocytes and Monocytes is described and illustrated in Supplementary Figure 1.

Single-cell suspensions from human peripheral blood mononuclear cells and CSF cells were stained for 30 min at 4 °C with the appropriate combination of indicated fluorescence-labeled monoclonal antibodies in PBS, containing 0.1% sodium azide and 0.1% bovine serum albumin (BSA) following treatment with VersaLyse (Beckman Coulter GmbH, Krefeld, Germany) according to the manufacturer’s instructions. The following monoclonal antibodies were used at 1:200 dilutions: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter). Flow cytometric analysis of stained cells was performed following standard protocols. Cells were analyzed on a Navios™ flow cytometer (Beckman Coulter) using Kaluza Analysis Software (V2.1, Beckman Coulter) and presented using Prism 6.0 (Graph Pad).

Human monocytes were derived from healthy donor buffy coats (Ethical Approval Number BTL 10.090) following Ficoll density gradient centrifugation and isolated from the PB mononuclear cell fraction using a negative selection cocktail to isolate unlabelled monocytes (StemCell Technologies, Grenoble, France). Purified monocytes were subsequently incubated with 1 μg ml⁻¹ CD14-FITC and 1 μg ml⁻¹ CD16-Pc5 (Beckman Coulter, Woerden, The Netherlands) for 30 min at 4 °C and FACS sorted using a FACSCalibur (BD Biosciences, Breda, The Netherlands). RNA was isolated from the subpopulations using Trizol reagent (Thermo Fisher Scientific, Bleiswijk, The Netherlands).

iPSC-derived endothelial cells were detached with trypsin and immunophenotyping was performed using the following monoclonal antibodies: CD31-FITC (1:25, BD Pharmingen, clone WM59), CD144-PE (1:10, Beckman Coulter, A07481), anti-KDR-APC (1:5, R&D Systems, clone 89106). CB-ECFCs phenotype was assessed with the same antibodies with additional negative controls CD 45-FITC (1:50, Beckman Coulter clone J33) and CD14-FITC (1:50, Beckman Coulter, clone RMO52). Antibodies and matched isotype control (Beckman Coulter) were incubated for 30 min at 4°C. Viability was assessed with 7-AAD (Becton Dickinson). Data were acquired and analyzed on a five-parameter flow cytometer (FACScalibur, Becton Dickinson, San Jose, CA) with Weasel software (WEHI, Melbourne, Australia).

On day 19 of cultures, the methylcellulose cultures were washed with PBS. Living cells were counted in trypan blue and stained with the following antibodies in PBS 4%BSA at 4 °C 45 min: CD45Pe-Vio770 (Miltenyi), CD34Vioblue (Miltenyi), CD41aPE (BD), CD43FITC (Miltenyi), CD31FITC (Beckman Coulter), CD235aAPC (BD), IL1-RAP APC (R&D), CD38PE (Miltenyi), CD71PE (BD), CD14FITC (Beckman Coulter), CD33APC (BD), CD133APC (BD), BB9PE (BD), SSEA1PE (BD). After 1 h, cells were washed and resuspended in PBS 4%BSA with viability staining reagent 1 µg/mL (7-AAD) 7-aminoactinomycin D (Sigma Aldrich). Stained cells were analyzed with a MACSQuant 10 (Miltenyi Biotec) flow cytometer and Flowjo analysis software.