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Stemflow hmsc analysis kit

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The BD Stemflow™ hMSC Analysis Kit is a laboratory equipment product designed for the analysis of human mesenchymal stem cells (hMSCs). It provides tools and reagents for the identification and characterization of hMSCs.

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Lab products found in correlation

Facscanto 2, by BD (6 mentions) Oil red o, by Merck Group (4 mentions) Facsdiva software, by BD (3 mentions) Alizarin red, by Merck Group (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Facsverse, by BD (2 mentions) Facsverse flow cytometer, by BD (2 mentions) Facscalibur, by BD (2 mentions) Phosphate buffered saline (pbs), by Corning (1 mentions) Stempro osteogenesis differentiation media, by Thermo Fisher Scientific (1 mentions) Stempro adipogenesis differentiation media, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Lsrfortessa cell analyzer, by BD (1 mentions) Facsaria, by BD (1 mentions) Cellquest pro software, by BD (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Cytoflex flow cytometer, by Beckman Coulter (1 mentions) Cytexpert, by Beckman Coulter (1 mentions) Caspase 3 antibody, by BD (1 mentions) Sc 5279, by Santa Cruz Biotechnology (1 mentions)

39 protocols using stemflow hmsc analysis kit

1

hMSC Immunophenotyping with BD Stemflow

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The BD StemflowTM hMSC Analysis kit (BD Biosciences) was used to identify the immunophenotype. First generation cells were cultured in a culture dish (10×1×10 cm). When the cell density reached 90%, the cells were washed with PBS (Corning, Inc.) twice and digested by trypsinase and then a single cell suspension was prepared. The cells were then separated into sterile tubes. A mesenchymal stem cell (MSC)-positive cocktail (CD90 FITC, CD105 PerCP-Cy5.5, CD73 APC, CD44) and MSC-negative cocktail (CD34, CD11b, CD19, CD45, HLA-DR) were added to the tubes in the dark at 4°C for 20 min. After the cells were identified by flow cytometry (BD Biosciences), PDLSCs were passaged to the third generation, and cells of this generation were used in the subsequent experiments.
Zhao B., Zhang Y., Xiong Y, & Xu X. (2019). Rutin promotes the formation and osteogenic differentiation of human periodontal ligament stem cell sheets in vitro. International Journal of Molecular Medicine, 44(6), 2289-2297.
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2

Characterizing Mesenchymal Stem Cells from Periodontal Ligament

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To identify the cell phenotype of PDLSCs, BD StemflowTMhMSC Analysis Kit (BD Biosciences, California, USA) was implemented according to the manufacturer’s instructions. For multilineage differentiation assays, PDLSCs were cultivated in 6-well plates at 2 × 105 cells per well. At 80%–90% density, the corresponding differentiation medium was replaced to assess osteogenesis and adipogenesis. After 21 days, the cells were stained by Alizarin Red (Sigma‐Aldrich, Missouri, USA) to observe the mineralization. After 28 days, the adipogenesis was detected by Oil Red O (Sigma) staining.
Wang Y., Wang L., Sun T., Shen S., Li Z., Ma X., Gu X., Zhang X., Peng A., Xu X, & Feng Q. (2023). Study of the inflammatory activating process in the early stage of Fusobacterium nucleatum infected PDLSCs. International Journal of Oral Science, 15, 8.
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3

Multipotent Mesenchymal Stem Cell Characterization

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Expanded MSCs were phenotypically tested for MSC markers. Briefly, a total of 0.25 × 10 5 cells were resuspended in 0.5 mL phosphate-buffered saline (Gibco, USA) and incubated with the antibodies of BD Stemflow TM hMSC Analysis Kit (BD Biosciences, USA) for 20 min at room temperature. The fluorescence intensity of the cells was evaluated by flow cytometry (BD FACSCanto II; BD Biosciences, USA).
Additionally, MSCs were tested for multilineage differentiation potential. For osteogenic and adipogenic differentiation, cells were cultured in StemPro osteogenesis differentiation media and Stem-Pro adipogenesis differentiation media (ThermoFisher, USA), respectively, as per the manufacturer's recommendation. Following osteogenic induction, cultures were stained using Alizarin Red (Allied Signal, Germany), and following adipogenic differentiation, cultures were stained using Oil Red O (Sigma-Aldrich, USA) to assess extracellular deposits.
Al Demour S., Adwan S., Jafar H., Rahmeh R., Alhawari H, & Awidi A. (2021). Safety and Efficacy of 2 Intracavernous Injections of Allogeneic Wharton's Jelly-Derived Mesenchymal Stem Cells in Diabetic Patients with Erectile Dysfunction: Phase 1/2 Clinical Trial. Urologia internationalis, 105(11-12).
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4

Immunophenotyping of Mesenchymal Stem Cells

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The immunophenotype of the MSCs was analyzed using flow cytometry. The cells were detached from the flask using trypsin, then centrifuged at 1500 rpm for 5 min, and resuspended in staining/wash buffer [phosphate-buffered saline (PBS) with 2% FBS)]. For staining, the BM-MSCs and HGF-eMSCs were washed with staining/wash buffer and incubated for 30 min in the dark at 4°C with Stemflow hMSC Analysis Kit (BD) according to the manufacturer’s instructions. The MSCs were characterized using the following conjugated monoclonal antibodies: CD90 fluorescein isothiocyanate (FITC) (Clone: 5E10), CD105 PerCP-Cy5.5 (Clone: 266), CD73 allophycocyanin (APC) (Clone: AD2), and phycoerythrin (PE) negative cocktail (CD34, CD11b, CD19, CD45, and HLA-DR). Flow cytometry was conducted using an LSRFortessa cell analyzer (BD Biosciences) and analyzed using FlowJo version 10 or BD FACSDiva software.
Park B.W., Jung S.H., Das S., Lee S.M., Park J.H., Kim H., Hwang J.W., Lee S., Kim H.J., Kim H.Y., Jung S., Cho D.W., Jang J., Ban K, & Park H.J. (2020). In vivo priming of human mesenchymal stem cells with hepatocyte growth factor–engineered mesenchymal stem cells promotes therapeutic potential for cardiac repair. Science Advances, 6(13), eaay6994.
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5

Flow Cytometry Analysis of MSC Markers

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Flow cytometry assay was performed to evaluate the expression of the MSCs markers. Stemflow hMSC Analysis Kit (BD Biosciences, Franklin Lakes, NJ, USA), was applied for flow cytometry assay, containing CD90 FITC (5E10), CD105 PerCP-Cy5.5 (266), CD73 APC (AD2), negative markers cocktail (CD45/CD34/CD11b/ CD19/HLA-DR-PE) and isotype controls. 1 × 106 ADSCs cells were analyzed by using flow cytometer (FACSAria; BD Biosciences).
Shen C., Quan Q., Yang C., Wen Y, & Li H. (2018). Histone demethylase JMJD6 regulates cellular migration and proliferation in adipose-derived mesenchymal stem cells. Stem Cell Research & Therapy, 9, 212.
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6

Immunophenotyping of Human Mesenchymal Stem Cells

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hMSC from passages 2 and 3 were dissociated using TrypLETM Select and suspended at 1×107 cells/mL in PBS-ethylenediaminetetraacetic acid (EDTA). Immunophenotypic analysis was performed using Stemflow hMSC Analysis Kit (BD, catalog #562245, USA) following manufacturer instructions, see Table S2. Briefly, 100 µL of cell suspension was incubated for 30 min at room temperature with a mix of antibodies positive for hMSC (CD90 FITC, CD105 PerCP-Cy5.5, CD73 APC), a negative cocktail of antibodies (CD34 PE/CD11b PE/CD19 PE/CD45 PE/HLA-DR PE), as well as isotype positive and negative control cocktails. After incubation, cells were washed with PBS-EDTA supplemented with 1% FBS and 0.09% of sodium azide, centrifuged and suspended in PBS for flow cytometry analysis. Samples were acquired using a FACSCalibur flow cytometer (BD, USA) and data analyzed with CellQuest Pro software (BD, USA).
Echarte L., Sujanov A., Machin D., Marquisá N, & Touriño C. (2023). Femur bone marrow from brain death deceased donors as source of human mesenchymal stromal cells for cell therapy. Stem Cell Investigation, 10, 12.
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7

Mesenchymal Stem Cell Surface Marker Profiling

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Cells were stained for CD73, CD90 and CD105, which have been regarded as positive surface antigens in mesenchymal stem cells (MSCs) [29 (link),30 (link)]. In contrast, CD34 which is expressed in leukocytes, and CD45 which is expressed in T and B lymphocytes, have been regarded as negative surface antigens in MSCs [31 (link),32 (link)]. Amplified cells at passage 4 were identified using flow cytometry with a Stemflow™ hMSC Analysis Kit (BD Biosciences, San Jose, CA). 1 ​× ​106 ​cells of each cell type were detached using detachment solution (1 ​× ​PBS, 1% FBS). Wash cells and resuspend at a concentration of 1 ​× ​107 ​cells/ml with similar staining buffer, then stained with CD73, CD90, CD105, CD34, or CD45 antibodies in bovine serum albumin stain buffer. Incubate in the dark for 30 ​min, wash the cells twice and resuspend at 300–500 ​μl in Stain Buffer (1 ​× ​PBS, 1% FBS). The stained cells were then analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Inc., USA), and analysis was performed using CytExpert (Beckman Coulter, Inc, USA).
Wu H., Tan J., Sun D., Wang X., Shen J., Wang S., Dai Q., Wei Z., Li G., Lin S., Luo F, & Xie Z. (2023). Discovery of multipotent progenitor cells from human induced membrane: Equivalent to periosteum-derived stem cells in bone regeneration. Journal of Orthopaedic Translation, 42, 82-93.
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8

Multiparametric Analysis of Cell Markers

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For quantification of γH2AX, cells were washed with PBS and fixed with ice-cold 70% ethanol. After washing with PBS, cells were incubated in incubation buffer (0.5% BSA/0.25% Triton X-100 in PBS) for 15 minutes on ice and incubated with Alexa Fluor 488 conjugated γH2AX antibody (BD Biosciences: 560445) or an isotype control (BD Biosciences: 557702) according to the manufacturer’s protocol. Samples were washed with incubation buffer, resuspended in PBS, and analyzed by BD FACS Canto II using FlowJo software. A minimum of 20,000 cells within the gated region were analyzed.
For characterization of iPSCs for cell surface markers (SSEA-4, TRA-1–60, and TRA-1–81), the following antibodies were used, such as Alexa Fluor 647 Mouse anti-SSEA-4, 560796; Alexa Fluor 488 Mouse anti-Human TRA-1–60, 560173; Alexa Fluor 647 Mouse anti-Human TRA-1–81, 560124 (BD Biosciences). For detection of intracellular marker (Oct-¾), cells were fixed in 2% paraformaldehyde, permeabilized in 0.1% Triton X-100 on ice, incubated with Oct-¾ antibody (1:20; sc-5279; Santa Cruz) for 1 hour at room temperature. MSCs were characterized using Stemflow hMSC analysis kit (BD Biosciences; 562245).
For EdU flow, cells were labeled with 10 uM EdU for 30 minutes and analyzed using Click-iT assay kit (ThermoFisher Scientific). Apoptosis was analyzed using caspase-3 antibody (BD Biosciences; 559585).
Vallabhaneni H., Lynch P.J., Chen G., Park K., Liu Y., Goehe R., Mallon B.S., Boehm M, & Hursh D.A. (2018). High Basal Levels of γH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair. Stem cells (Dayton, Ohio), 36(10), 1501-1513.
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9

Evaluating Adipogenic and Osteogenic Potential of MSCs

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To test their adipogenic differentiation potential, MSCs were cultured in medium consisting of DMEM high glucose media with 10 % (v/v) FCS, 2 mM L-glutamine, 100 U/ml Pen/Strep, 1 mM dexamethason (Sigma), 0.5 mM 1-methyl-3-isobutylxanthin (Sigma) and 10 mg/ml Insulin (Sigma). Medium was changed twice a week. Osteogenic differentiation was induced using DMEM low glucose with 10 % (v/v) FCS, 2 mM L-glutamine, 100 U/ml Pen/Strep, 100 nM dexamethason (Sigma), 200 mM L-ascorbic acid 2-phosphate (Sigma) and 10 mM B-glycerophosphate (Sigma). After 3 weeks the cells were stained with Oil red O (Sigma) for adipogenic and Alizarin red S (Sigma) for osteogenic differentiation. To check for the MSC-specific immunophenotype, surface marker expression was tested using the Stemflow hMSC Analysis Kit (BD Biosciences).
Erdmann G., Suchanek M., Horn P., Graf F., Volz C., Horn T., Zhang X., Wagner W., Ho A.D, & Boutros M. (2015). Functional fingerprinting of human mesenchymal stem cells using high-throughput RNAi screening. Genome Medicine, 7(1), 46.
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10

Characterizing hUC-MSC Surface Markers

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The hUC-MSCs surface markers, i.e. CD105, CD90 and CD73, were detected by flow cytometry using BD Stemflow hMSC Analysis Kit following the procedures described previously [8 (link), 27 (link)].
Na T., Zhang K, & Yuan B.Z. (2020). The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stromal /stem cells through interacting with the Notch1 protein. BMC Cancer, 20, 1064.
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