Murine ADSC were obtained from the abdominal fat of C57BL/6 mice, as previously described [30 (link)]. ADSC were cultured in EGM-2 media (Lonza, Basel, Switzerland), consisting of 10% FBS (Gibco, MD, USA) and 1% penicillin/streptomycin (Gibco), 2 mM L-glutamine (Corning Life Sciences, Corning, NY, USA) on plastic dishes at 37°C in 5% CO2. Cell passages 2–6 were used in this study. ADSC identity was confirmed by FACS analysis of passage 2 cells [30 (link)]. ADSC identity was verified using anti-CD34, anti-CD90, anti-Sca-1, anti-CD105, anti-CD34 and anti-CD44 (BioLegend, CA, USA), using an LSRII flow cytometer (BD Biosciences, CA, USA).
Anti cd34
Manufactured by BioLegend
Sourced in United States
Anti-CD34 is a monoclonal antibody used to detect the CD34 protein, which is expressed on the surface of hematopoietic stem and progenitor cells. This antibody can be used in various research applications, such as flow cytometry and immunohistochemistry, to identify and study CD34-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45, by BioLegend (12 mentions)
Anti cd90, by BioLegend (12 mentions)
Anti cd105, by BioLegend (10 mentions)
Anti cd73, by BioLegend (8 mentions)
Anti cd19, by BioLegend (6 mentions)
Anti cd44, by BioLegend (5 mentions)
Anti cd29, by BioLegend (4 mentions)
Anti hla dr, by BioLegend (4 mentions)
Anti cd11b, by BioLegend (4 mentions)
Anti cd31, by BioLegend (4 mentions)
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
Anti cd10, by BioLegend (2 mentions)
Anti cd45ra, by BioLegend (2 mentions)
Anti cd45, by BD (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Facscanto 2, by BD (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by Bio-Rad (1 mentions)
Anti pd 1, by BioLegend (1 mentions)
Anti cd86, by BioLegend (1 mentions)
26 protocols using anti cd34
1
Isolation and Characterization of Murine Adipose-Derived Stem Cells
2
Flow Cytometric Analysis of BM-MNC
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To 100 μl of BM-MNC suspension in physiological saline (8.8 × 106 cells/ml), FITC-labeled anti-CD14 (Serotec, Oxford, UK); anti-CD90 (Serotec); anti-CD4 (Serotec) antibodies; and anti-CD34 (Biolegend, San Diego, CA) were added, and the mixture was incubated for 30 minutes at room temperature (RT). After incubation, 900 μl of phosphate-buffered saline was added and mixed thoroughly. Cell compositions were analyzed using an Accuri C6 flow cytometer (Accuri Cytometers, Inc., Ann Arbor, MI), 100,000 events per sample. Cell viability was determined using the 7-AAD method. The number of transplanted cells is shown in Table 2 .
3
Purification and Characterization of B Cells
4
Purification and Transduction of Primitive Hematopoietic Cells
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Cells were labeled with anti-human LIN cocktail (anti-CD3/CD14/ CD16/CD19/CD20/CD56), anti-CD15, anti-CD34, anti-CD38, anti-CD45RA (Biolegend) and anti-CD90 (BD Biosciences), antibodies specification described above, to perform purification of Primitive subpopulations (Lin- CD34+CD38- CD45RA- cells). The stained samples were FACS purified with the BD Aria cell sorter (BD Biosciences), achieving purity ranging between 92% and 99%.
Sorted primitive (HSC+MPP) populations were transduced as described above in “In vitro transduction of HD and WAS patients’ derived BM or MPB CD34+ cells” section. After transduction, cells were collected, washed, and transplanted in NSGW41 mice. An aliquot of cells was cultured in Iscove’s modified Dulbecco’s medium (IMDM), 10% fetal bovine serum (Cambrex, East Rutherford, NJ, USA) with stem cell factor (SCF), FLT3-L thrombopoietin (TPO) and IL-3 (all from Preprotech) at 20 ng/ml concentration (liquid culture) and harvested after 15 days to perform VCN estimation. An additional aliquot was used to perform Colony Forming Cell (CFC) assay according to the manufacturer’s procedure in Methocult medium (Stem Cell Technologies, Vancouver, Canada). At day 14, colonies were scored, singly picked and analyzed to evaluate the percentage of transduction.
Sorted primitive (HSC+MPP) populations were transduced as described above in “In vitro transduction of HD and WAS patients’ derived BM or MPB CD34+ cells” section. After transduction, cells were collected, washed, and transplanted in NSGW41 mice. An aliquot of cells was cultured in Iscove’s modified Dulbecco’s medium (IMDM), 10% fetal bovine serum (Cambrex, East Rutherford, NJ, USA) with stem cell factor (SCF), FLT3-L thrombopoietin (TPO) and IL-3 (all from Preprotech) at 20 ng/ml concentration (liquid culture) and harvested after 15 days to perform VCN estimation. An additional aliquot was used to perform Colony Forming Cell (CFC) assay according to the manufacturer’s procedure in Methocult medium (Stem Cell Technologies, Vancouver, Canada). At day 14, colonies were scored, singly picked and analyzed to evaluate the percentage of transduction.
5
Characterization of hucMSC Surface Markers and Differentiation
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To evaluate the surface markers of hucMSCs, hucMSCs at passage three were incubated with antibodies anti-CD34 (catalog number: 343503, BioLegend, San Diego, CA, USA), anti-CD73 (catalog number: 344015, BioLegend), anti-CD29 (catalog number: 303004, BioLegend), anti-CD14 (catalog number: 397706, BioLegend), anti-CD105 (catalog number: 323203, BioLegend), anti-CD19 (catalog number: 302205, BioLegend), anti-CD45 (catalog number: 304005, BioLegend), anti-HLA-DR (catalog number: 327005, BioLegend), and anti-CD90 (catalog number: 328108, BioLegend). Fluorescence was detected using a flow cytometer (NovoCyte 1300; ACEA, San Diego, CA, USA) to identify hucMSCs.
To evaluate cell differentiation, hucMSCs at passage three were cultured in adipogenic (catalog number: A1007001, Gibco, Grand Island, USA), chondrogenic (catalog number: A1007101, Gibco), or osteogenic medium (catalog number: A1007201, Gibco) for 3 weeks. Subsequently, the cells were fixed with 4% paraformaldehyde, and stained with oil red O (catalog number: HY-D1168, MedChemExpress, New Jersey, USA), alcian blue (catalog number: HY-D0001, MedChemExpres), or alizarin red (catalog number: HY-120601, MedChemExpres), respectively. The stained cells were observed under a light microscope (Olympus, Tokyo, Japan).
To evaluate cell differentiation, hucMSCs at passage three were cultured in adipogenic (catalog number: A1007001, Gibco, Grand Island, USA), chondrogenic (catalog number: A1007101, Gibco), or osteogenic medium (catalog number: A1007201, Gibco) for 3 weeks. Subsequently, the cells were fixed with 4% paraformaldehyde, and stained with oil red O (catalog number: HY-D1168, MedChemExpress, New Jersey, USA), alcian blue (catalog number: HY-D0001, MedChemExpres), or alizarin red (catalog number: HY-120601, MedChemExpres), respectively. The stained cells were observed under a light microscope (Olympus, Tokyo, Japan).
6
Multiparameter Immune Cell Profiling
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Anti-mouse ABs: anti-CD3e (clone: 145-2C11), anti-CD34 (clone: HM34), anti-CD45R (clone: RA3-6B2), anti-CD117 (c-KIT, clone: 2B8), anti-CD182 (CXCR2, clone: SA044G4), anti-CD184 (CXCR4, clone: L276F12), anti-Ly-6A/E (Sca-1, clone: D7), anti-Ki-67 (clone: 11F6), anti-Ly-6G (clone: 1A8), anti-NK-1.1 (clone: PK1), anti-mouse/human-CD11b (clone: M1/70) all from BioLegend (San Diego, CA, USA), and anti-CD16/32 (clone: 93) and anti-CD62L (clone: MEL-14) (both from eBioscience, Thermo Fisher Scientific, Waltham, MA, USA). Anti-human ABs: anti-CD66b (clone: 80H3, Beckman Coulter, Brea, CA, USA), and anti-CD71 (clone: M-A712, BD Biosciences, Franklin Lakes, NJ, USA). Viability dye eFluor780 and eFluor506 (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) or DAPI (BioLegend, San Diego, CA, USA) were used to determine viable cells.
7
Characterization of GMP-Grade Adipose MSCs
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Good Manufacturing Practices (GMP)-Grade human adipose-derived MSCs (Steminent Biotherapeutics Inc., Taiwan) were cultured in MSC maintenance medium consisting of IMDM, 10% FBS (#10270106, Gibco®, Thermo Fisher Scientific, Waltham, MA, USA), 10 ng/mL bFGF (#233-FB, R&D Systems, Minneapolis, MN, USA) and 1% PSG (#10378016, Gibco®). Human skin fibroblasts (#GM08429, Coriell Institute, Camden, NJ, USA) were cultured in alpha MEM supplemented with 15% FBS and 1% PSG at a seeding density of 3000 cells/cm2, and the cells were subcultured after reaching confluence. MSC characteristics were confirmed according to the minimal criteria defined by the International Society for Cell and Gene Therapy (ISCT) [35] (link). Surface markers were analyzed using a FACSAria Fusion Cell sorter and Cell Analyzer (BD Biosciences, San Jose, CA, USA). Primary antibodies for flow cytometry were as follows: anti-CD105, anti-CD90, anti-CD73, anti-CD34, anti-CD45 and anti-CD11b (#800505, #328107, #344015, #343607, #368511, #301309, BioLegend, San Diego, CA, USA; 1:100 dilution). Multidifferentiation capacities were assessed by alkaline phosphatase and Oil Red O, as previously described [9] (link), [36] (link).
8
Immunophenotyping of Mesenchymal and Immune Cells
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Flow cytometric analyses were performed with Gallios (Beckman Coulter) flow cytometers, and the data were analyzed with the FlowJo (Treestar) software packages.
MSCs were incubated with anti-CD90, anti-CD105, anti-CD73, anti-CD44, anti-CD11B, anti-CD34, anti-CD19, anti-CD45 and anti-HLA-DR antibodies to examine MSC surface markers, which were purchased from BioLegend. Anti-CD3, anti-CD4, anti-CD8, anti-TNF-α and anti-IFN-γ antibodies were used to examine stain T cells. Propidiumiodide (PI; BD) and Annexin V (BD) were used to stain apoptosis cells. Anti-CD86, anti-F4/80 and anti-CD45 antibodies were used to stain macrophages.
MSCs were incubated with anti-CD90, anti-CD105, anti-CD73, anti-CD44, anti-CD11B, anti-CD34, anti-CD19, anti-CD45 and anti-HLA-DR antibodies to examine MSC surface markers, which were purchased from BioLegend. Anti-CD3, anti-CD4, anti-CD8, anti-TNF-α and anti-IFN-γ antibodies were used to examine stain T cells. Propidiumiodide (PI; BD) and Annexin V (BD) were used to stain apoptosis cells. Anti-CD86, anti-F4/80 and anti-CD45 antibodies were used to stain macrophages.
9
Immunophenotypic Characterization of Cells
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For cell surface marker staining, cells were collected, washed once with PBS supplemented with 2% FCS (FACS buffer). Cells were incubated with anti CD34 (#343516581), CD90 (#3281145E10) and EPCR (#351906) (BioLegend) antibodies for 30 minutes (min) at 4°C, and washed once with cold FACS buffer. For cell sorting, CD34+ cells were quickly thawed and stained for CD34, CD38 (#345806), CD45RA (#560362) (BD Bioscience) and CD90 following the same procedure as above. When specified, cells were stained with the Annexin V Apoptosis Detection Kit, according to the manufacturer’s protocol (BD Bioscience). All data were collected on FACS Canto II or LSRII analyzer (Becton Dickinson), and analyzed with FlowJo software. Cells were sorted on a FACS Aria II or III (Becton Dickinson).
10
Identification and Characterization of Circulating Tumor Cells
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CTCs were assessed by immunofluorescence staining. The following antibodies were used to identify CTCs and assess EPCAM and TROP-2 staining as indicated in the figure legends: Anti-CD45 (BioLegend Cat# 304018, RRID:AB_389336), Anti-CD34 (BioLegend Cat# 343508, RRID:AB_1877133), Anti-CD11B (BioLegend Cat# 101218, RRID:AB_389327), Anti-CD66B (BioLegend Cat# 305109, RRID:AB_2563170), Anti-Pan cytokeratin (BioLegend Cat# 628602, RRID:AB_439775 or Abcam Cat# ab49779, RRID:AB_869395), Anti-Androgen Receptor (Cell Signaling Technology Cat# 5153S, RRID:AB_10692774), Anti-EPCAM (Abcam Cat# ab112068, RRID:AB_10861805) or Anti-TROP-2 (BD Biosciences Cat# 940370, RRID:AB_2876239) and Hoechst 33342 (Thermo Fisher Scientific). Extracellular antibodies were stained at 4°C for 30 minutes. For intracellular and nuclear staining of cells (Fig. 2 ), cells were stained as described by Sperger and colleagues (30 (link)). For intracellular staining (Fig. 4 ), cells were permeabilized, stained, and washed with BD Perm/Wash. Images were taken with a 10x objective using Nikon Eclipse Ti-E with an ORCA-Flash 4.0 V2 Digital CMOS camera (Hamamatsu) and NIS-Elements AR Microscope Imaging Software (RRID:SCR_014329, Nikon Instruments). Images were background subtracted, and CTCs were determined by Hoechst-positive staining, cytokeratin+ and CD45−/CD34−/CD66b−.
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