Stemflow human msc analysis kit

The flow cytometry methods employed here are identical to those described in our adjacent paper [17 ] to permit comparisons between the results of MSCs expanded in static culture and those expanded in dynamic culture. Briefly, the BD Stemflow™ Human MSC Analysis Kit (BD Biosciences, Cat. number 562245) was used for positive and negative surface marker staining. Using the manufacturer's protocol, hUC MSC samples were stained with four fluorochromes together including positive and negative staining cocktails. The positive marker cocktail stained for CD90, CD105, and CD73 (defined as >97% positively stained cells). The negative cocktail (all antibodies were stained using a single fluorochrome, PE) stained for CD34, CD45, CD11b, CD19, and HLA-DR (defined as <2% positively stained cells). A CD44 labeled PE antibody was used as positive control for the negative cocktail to set the compensation and gating of the negative cocktail. For additional methodological details, see [17 ].

To analyze the expression of cluster of differentiation (CD) markers typical for MSCs on the surface of hADSCs and isolate from them CIMVs, the following antibodies were used: CD90 (FITC) (#555595, BD Biosciences, San Jose, CA, USA), CD29 (APC) (#303008, BioLegend, San Diego, CA, USA), CD166 (PE) (#343904, BioLegend, San Diego, CA, USA), CD44 (PE) (#103024, BioLegend, San Diego, CA, USA), CD73 (APC) (#344006, BioLegend, San Diego, CA, USA), and a negative control (PE) (BD Stemflow™ Human MSC Analysis Kit, BD Biosciences, San Jose, CA, USA) including antibodies to CD34, CD11b, CD19, CD45, and HLA-DR. Briefly, 1 × 10⁵ native hADSCs, hADSCs-BFP and hADSCs-IL2 were trypsinized and washed two times with PBS, and stained with the antibodies for 30 min in the dark at room temperature. Cells were washed once with PBS and analyzed by flow cytometry using FACSAria III (BD Biosciences, San Jose, CA, USA), and data were analyzed using BD FACSDiva™ software version 7.0.
CIMVs were isolated from 2 × 10⁵ native hADSCs, hADSCs-BFP and hADSCs-IL2, washed once with PBS and stained with the listed above antibodies for 30 min in the dark at room temperature. Then, CIMVs were washed once with PBS and analyzed using the FACSAria III flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using BD FACSDiva™ software version 7.0.

hBM-MSCs were characterized at P3-P4 and P6-P7 by flow cytometry using antibodies to CD44, CD73, CD90, and CD105 cell surface markers and using a mixture of antibodies to CD34, CD11b, CD19, CD45, HLA-DR cell surface markers (BD Stemflow™ Human MSC Analysis Kit). After harvesting, the cells were washed with PBS and treated according to the manufacturer's protocol. Viability of the MSCs samples was assessed by 5-minute 7-AAD staining and cell granularity was determined by side-scattered (SSC) light evaluation. Cytometric measurements were performed on BD LSR II flow cytometer. 10.000 cells per tube were analyzed with FlowJo X software.

Characterisation of the WJMSCs was performed based on their morphology, surface marker, and differentiation ability. Immunophenotyping analysis to detect the surface marker of the WJMSC was conducted according to the manufacturer protocol using a BD Stemflow Human MSC Analysis Kit (BD Biosciences, San Jose, CA, USA). The differentiation ability of WJMSCs was performed using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.

The isolated MSCs were characterized by phenotyping with MSC-specific cell surface markers using the BD Stemflow-Human MSC Analysis Kit (BD Biosciences, Franklin Lakes, NJ, United States), according to the International Society for Cellular Therapy guidelines (23 (link)). Flow cytometry was performed using a fluorescence-activated cell sorter (BD FACS Aria II, BD Biosciences, Franklin Lakes, NJ, United States), and the results were analyzed with DIVA software. The obtained MSCs fulfilled the International Society for Cellular Therapy criteria (23 (link)), including a phenotypic characterization: CD105/CD73/CD90/CD44 positive and CD45/CD34/CD11b/CD19/HLA-DR PE negative.