CD45-APC (BD, San Jose, CA, USA; 555485), CD33-PE (BD 555450), CD44-FITC (BD 555478), CD44-PE-Cy7 (BD 560533), CD45.1-PerCP-Cy5.5 (BD 560580), CD34-APC-Cy7 (Biolegend, Ozyme, Saint Quentin Yvelines, France; 343514), Lin1-FITC (BD 340546), CD123-PE (BD 340545), CD45RA-Alexa Fluor 700 (BD 560673) and CD38-APC (BD 555362) fluorescent antibodies were used to analyze leukemic cells before and after injection into animals to determine phenotypic analysis of engrafted cells and percentage of leukemic cell engraftment. Absolute cell counts were determined with CountBright absolute counting beads (Invitrogen) following the manufacturer's recommendations.
Cd34 apc cy7
Manufactured by BioLegend
Sourced in France, United States, United Kingdom
The CD34 APC-Cy7 is a fluorescent-conjugated antibody that binds to the CD34 antigen, which is a marker for hematopoietic stem and progenitor cells. It can be used in flow cytometry applications to identify and quantify CD34-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd90 fitc, by BioLegend (3 mentions)
Cd45 apc, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Cd31 fitc, by BioLegend (2 mentions)
Cd11b apc, by BioLegend (2 mentions)
Cd146 pe, by BioLegend (2 mentions)
Cd73 apc, by BioLegend (2 mentions)
Vegfr2 pe, by BioLegend (2 mentions)
Cd45 bv605, by BioLegend (2 mentions)
Cd33 pe, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Cd123 pe, by BD (1 mentions)
Cd38 apc, by BD (1 mentions)
Cd44 pe cy7, by BD (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Cd45 1 percp cy5, by BD (1 mentions)
Lin1 fitc, by BD (1 mentions)
Cd45ra alexa fluor 700, by BD (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
10 protocols using cd34 apc cy7
1
Immunophenotypic Analysis of Leukemic Engraftment
2
Isolation and Sorting of AML Stem Cells
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8227 cells were stained with CD34–APC-Cy7 (BioLegend) and CD38–PE-Cy7 (BD Biosciences) and washed with PBS before sorting CD34+CD38− and CD34−CD38+ cells. Cells were FACS-sorted on a MoFlo system (Beckman Coulter). Primary AML specimens were stained with BB515–CD45 (BD Biosciences, 564585) to identify the blast population, PE–CD19 (BD Biosciences, 555413) and PE-Cy7–CD3 (BD Biosciences, 557749) to exclude the lymphocyte populations, 4,6-diamidino-2-phenylindole (DAPI; EMD Millipore, 278298) as a dead-cell marker and CellROX deep red (Thermo Fisher Scientific, C10422), and sorted using a Sony SH-800 system. ROS-low LSCs were identified as the cells with the 20% lowest ROS levels and the ROS-high blasts were identified as the cells with the highest 20% ROS levels, as recently described in detail14 (link).
3
Characterization of Mesenchymal Stem Cells
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Samples from four women were used in this step. The cells were evaluated according to the criteria for characterization of MSCs33 (link). Regarding adhesion to polystyrene and formation of fibroblast-like forming colonies, immunophenotypic15 characterization was done by flow cytometry by labeling the cells with the following antibodies: CD73-PECy7, CD90-FITC, CD105- FITC, CD19-PE Cy7, CD34-APC Cy7, CD45-APC, HLA-DR-PERCP Cy5.5, CD44-PERCP Cy5.5, CD49c-PE, CD151-APC, CD166-PE, CD117–APC, SSEA4-FITC, Oct4-PE (Biolegend, San Diego, CA), and NANOG-PERCP Cy5.5 (BD Pharmingen). The analysis was done in BD FACSCanto or BD FACSVerse and the results were analyzed using BD FACSDiva.
Analysis of differentiation into the three main mesenchymal lines was performed using adipogenesis, chondrogenesis, and osteogenesis StemPro differentiation kits (Gibco), according to the manufacturer's protocol. 0.5% oil Red O (Sigma-Aldrich), Alcian blue (Sigma-Aldrich), and Alizarin red (Sigma-Aldrich) were used respectively to observe adipogenesis, chondrogenesis, and osteogenesis.
Analysis of differentiation into the three main mesenchymal lines was performed using adipogenesis, chondrogenesis, and osteogenesis StemPro differentiation kits (Gibco), according to the manufacturer's protocol. 0.5% oil Red O (Sigma-Aldrich), Alcian blue (Sigma-Aldrich), and Alizarin red (Sigma-Aldrich) were used respectively to observe adipogenesis, chondrogenesis, and osteogenesis.
4
Flow cytometric analysis of cell markers
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Cells were dissociated to form a single-cell suspension by TrypLE treatment and washed with FACS buffer PBE (2% FBS and 0.5 mM EDTA in PBS). The dissociated cells were then resuspended in PBE and labeled with fluorochrome-conjugated anti-human CD73-PE-Cy7 (BioLegend, clone: AD2), CD184-APC (Invitrogen, clone: 12G5), CD144-PE (Invitrogen, clone: 16B1), CD34-APC-Cy7 (BioLegend, clone: 561), CD34-PE (Invitrogen, clone: 4H11), and CD43-PE (BioLegend, clone: 10G7). Dead cells were excluded according to DAPI (BD Biosciences) staining. Isotype-matched control antibodies were used to determine the background. Flow cytometry was performed using Canto II analyzer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Inc.).
5
Phenotypical Analysis of Stromal Vascular Fraction
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The SVF cell phenotype was determined by 6-channel cytofluorimetric analysis before and after the culture. For this latter condition, constructs were digested in 0.15% w/v collagenase (Worthington Biochemical Corporation) and cells were collected by centrifugation. Cells in suspension were then incubated for 30 min on ice with different fluorochrome-conjugated antibodies to human CD90 FITC, CD73 APC, CD31 FITC, CD34 APC-Cy7, CD146 PE, CD45 BV605, VEGFR2 PE, and CD11b APC (all from Biolegend) in staining buffer (PBS, 0.5% v/v FBS, 2 mM EDTA). 5 µl per million cells were used for all the antibodies according to the manufacturer’s protocol, and data were acquired with LSRFortessaTM flow cytometer (BD Biosciences), analyzed using Flowjo v10.1r5 software (Tree Star), and presented as percentage over the live cells (DAPI positive) or ratio over the fresh SVF population.
6
Multiparametric flow cytometry analysis
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Bones and spleens were crushed, cells resuspended and filtered to obtain a single-cell suspension that was analyzed by flow cytometry on a FACSCanto (BD Biosciences). Cells were stained with the following antibodies: CD45-APC, CD10-BV605, CD15-BV605 and glycophorin A-PE (from BD Biosciences) and CD33-BV421, CD19-PerCPCy5.5, CD34-APCCy7, CD68-PE, CD14-BV605, CD117-PECy7, IgM-PE, CD3-PECy7, FceRI-PE and CD25-BV421 (from BioLegend, San Diego, CA, USA). Control cells were stained with matching isotype controls. Sorting of cells was performed on a FACSAria (BD Biosciences).
For intracellular staining of phosphorylated STAT5 (signal transducer and activator of transcription 5), sorted pre-B cells were fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were stored in 90% ethanol in −80 °C until analysis. Cells were washed two times in ice cold phosphate-buffered saline before resuspension in phosphate-buffered saline with 2% fetal calf serum. Cells were kept on ice and stained with antibodies against phosphorylated STAT5 (STAT5P-Alexa Flour 647 from BD) or matching isotype controls. Levels of phosphorylated STAT5 are presented as median fluorescence intensity, normalized to the median fluorescence intensity of isotype-stained cells.
For intracellular staining of phosphorylated STAT5 (signal transducer and activator of transcription 5), sorted pre-B cells were fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were stored in 90% ethanol in −80 °C until analysis. Cells were washed two times in ice cold phosphate-buffered saline before resuspension in phosphate-buffered saline with 2% fetal calf serum. Cells were kept on ice and stained with antibodies against phosphorylated STAT5 (STAT5P-Alexa Flour 647 from BD) or matching isotype controls. Levels of phosphorylated STAT5 are presented as median fluorescence intensity, normalized to the median fluorescence intensity of isotype-stained cells.
7
Flow Cytometry Immunophenotyping of HSPCs
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Flow cytometric immunophenotyping of HSPCs was done on stored frozen viable mononuclear cells (MNCs) from PB and/or BM samples obtained from individuals with SDS aged 4–33 years or from healthy/non-SDS donors aged 29–32 years (STEMCELL Technologies). MNCs from healthy (non-SDS) donors were stained with antibodies: CD3-FITC (clone HIT3a, BD #555339; dilution 1:500), CD90-PE (clone 5E10, Biolegend, #328114; 1:33), CD49f-PECy5 (clone GoH3, BD #551129; 1:100), CD38-PECy7 (clone HIT2, Biolegend, #303516; 1:100), CD33-APC (clone WM53, BD #571817; 1:200), CD19-A700 (clone HIB19, Biolegend #302226; 1:300), CD34-APCCy7 (clone 581, Biolegend #343514; 1:100), CD45RA-BV421 (clone HI100, Biolegend #304130; 1:100), and Zombie Aqua (Biolegend #423101; 1:2000). MNCs from individuals with SDS were stained with the following antibodies: CD38-FITC (clone HIT2, BD #555459; 1:12.5), CD34-PE-Cy7 (clone 8G12, BD #348811; 1:33), CD10-BV605 (clone HI10a, Biolegend #312222: 1:33), CD45RA-V450 (clone HI30, BD #560367; 1:100), CD90 APC (Clone 5E10, Biolegend #328110; 1:50), CD3-APC-Cy7 (clone SK7, Biolegend #344818; 1:50), and CD19-APC-Cy7 (clone HIB19, Biolegend #302218, 1:50). After gating for live singlets (7AAD or Zombie negative) and excluding CD3/CD19 positive cells, bulk CD34 positive progenitors were gated (Supplementary Fig. 1 ).
8
Screening Leukemia Stem Cell Inhibitors
9
Scaffold Cell Composition and Viability
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To assess the cellular composition and viability in the scaffolds, cells were stained with the DNA-intercalating viability dye 7-AAD, and the antibodies CD34-ApCCy7, CD38-APC, CD73-FITC and CD10-PE (all from BioLegend, London, UK). Briefly, cells were washed once (300×g, 5 min, 4 °C), resuspended in PBS-0.5% BSA and placed on ice. Antibody cocktails (4 colour and fluorescence minus one (FMO)) were prepared in the dark, 250 μL cell samples were transferred to a 96 well round bottom ULA plate (Corning) and stained with antibody cocktails at 4 °C for 30 min in the dark. Plates were washed twice (300×g, 5 min, 4 °C) and pellets resuspended in 250 μL fresh PBS-0.5% BSA before acquisition on the Guava easyCyte 8HT flow cytometer (Millipore, Burlington, MA, USA). Guava single stained beads were used as antibody compensation controls following manufacturer’s instructions (AbC total Antibody Compensation Bead Kit; Thermo Fischer Scientific, Waltham, MA, USA). Freeze-killed 3D model-harvested cells were used for the 7-AAD compensation control (stain dead cells), vehicle-treated cells were used for the unstained control. Data were analysed using FlowJo 10 (FlowJo LLC, Ashland, OR, USA).
10
Comprehensive Characterization of Freshly Isolated SVF Cells
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The characterization of freshly isolated SVF cells from three donors was performed by using 6-channel cytofluorimetric analyses. Cell suspension was incubated for 30 min on ice with different fluorochrome-conjugated antibodies to human CD90 FITC, CD73 APC, CD31 FITC, CD34 APC-Cy7, CD146 PE, CD45 BV605, VEGFR2 PE, and CD11b APC (all from Biolegend) in staining buffer (PBS, 0.5% v/v FBS, 2mM EDTA).
Stromal mesenchymal cells, progenitor and mature endothelial cells, and myeloid cells were identified as CD45 -CD73 + CD90 + , CD45 -CD31 + CD34 + , CD45 -CD31 + VEGFR2 + , and CD45 + CD11b + , respectively. Pericytes were identified as CD45 -CD34 - CD146 + , as previously described [31] . 5 µl per million cells were used for all the antibodies according to the manufacturer's protocol, and data were acquired with LSRFortessaTM flow cytometer (BD Biosciences), analyzed using Flowjo v10.1r5 software (Tree Star), and presented as percentage over the living cells, positive stained for (4',6-diamidino-2-phenylindole) DAPI (Invitrogen).
Stromal mesenchymal cells, progenitor and mature endothelial cells, and myeloid cells were identified as CD45 -CD73 + CD90 + , CD45 -CD31 + CD34 + , CD45 -CD31 + VEGFR2 + , and CD45 + CD11b + , respectively. Pericytes were identified as CD45 -CD34 - CD146 + , as previously described [31] . 5 µl per million cells were used for all the antibodies according to the manufacturer's protocol, and data were acquired with LSRFortessaTM flow cytometer (BD Biosciences), analyzed using Flowjo v10.1r5 software (Tree Star), and presented as percentage over the living cells, positive stained for (4',6-diamidino-2-phenylindole) DAPI (Invitrogen).
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