Human umbilical cord blood-derived CD34+ cells (purchased from Lonza or Stem Cell Technologies) or FACS-purified CD34+CD38-CD90+CD49f+ cells (following staining with APC-CD34 [Biolegend; 560940], PE-CD38 [BD; 347687], FITC-CD90 [Biolegend; 328113], and APC/Cy7-CD49f [Biolegend; 313611]) were cultured in IMDM (Life Technologies) containing 0.1% HSA or 0.1% PVA (Sigma P8136, 363081, or 363146), supplemented with 1% ITSX, 1% P/S/G, 10 mM HEPES. For proliferation assays, 50 cells were seeded per well and supplemented with 10 ng/ml human SCF and 100 ng/ml human TPO (Peprotech). During the cultures, media was refreshed every three days and counted at day-7. For xenograft assays, 2×103 cells were expanded for 7-days before being injected intravenously into sub-lethally-irradiated (1.5 Gy) NOG mice. Human cell chimerism in the PB was calculated at 16-weeks post-transplantation using PE/Cy7-CD45.1 (Biolegend; 110730) and V450-hCD45 antibodies (BD; 560367).
Cd34 apc
Manufactured by BioLegend
Sourced in United States
The CD34-APC is a fluorochrome-conjugated antibody that specifically binds to the CD34 antigen, which is a transmembrane phosphoglycoprotein expressed on hematopoietic stem and progenitor cells. This product is intended for use in flow cytometry applications to identify and quantify CD34-positive cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 2, by BD (6 mentions)
Cd90 fitc, by BioLegend (4 mentions)
Cd45 pe, by BioLegend (4 mentions)
Facscanto 2, by BD (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Cd45 pe cy7, by BioLegend (2 mentions)
Cd38 pe, by BD (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Human scf, by Thermo Fisher Scientific (2 mentions)
Apc cy7 cd49f, by BioLegend (2 mentions)
Human tpo, by Thermo Fisher Scientific (2 mentions)
V450 hcd45 antibodies, by BD (2 mentions)
Summit v5, by Beckman Coulter (2 mentions)
Percp cy5.5 cd3, by BioLegend (2 mentions)
Moflo xdp70, by Beckman Coulter (2 mentions)
Gamunex, by Baxter (2 mentions)
Apc cd151, by BioLegend (2 mentions)
Cd45 pacific blue, by BioLegend (2 mentions)
Pe cyanine7 cd38, by Thermo Fisher Scientific (2 mentions)
Cd45 percp, by BioLegend (2 mentions)
27 protocols using cd34 apc
1
Expansion and Xenotransplantation of Human Cord Blood CD34+ Cells
2
Immune Cell Subset Characterization
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Excised tissues were prepared as previously described (41 (link)). Cell suspensions were blocked by the addition of Gamunex (Baxter, Deerfield, IL) for 30 minutes at RT, then washed in flow buffer (PBS + 2%FBS) and incubated at RT for 1 hour with the following antibodies (at a 1:10 final concentration): PerCP/Cy5.5-CD3, APC-CD34, PE-CD45, and/or APC-CD151 (BioLegend). Flow analysis was conducted on a Beckman Coulter Cyan. Cell sorting was performed on the Beckman Coulter Moflo XDP 70. Data analysis was performed using Summit, V5.1 (Beckman Coulter, Brea, CA).
3
Immune Cell Subset Characterization
4
Isolation of CD34+ Cells from Frozen UCB
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Frozen UCB units were provided by the Japanese Red Cross Kanto-Koshinetsu Cord Blood Bank (Tokyo, Japan). Isolation of human CD34+ cells from frozen UCB samples starts with introduction of thawed UCB units into CliniMACS Prodigy equipment (Miltenyi Biotec). In a closed tube-bag system, UCB cells were washed in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) containing DNase I (Roche), followed by centrifugation (420 g). This medium was replaced by CliniMACS Buffer and MNCs were isolated from the entire unit in a gradient formed using Ficoll-Paque Premium buffer (GE Healthcare; P = 1.084). MNCs were then washed three times in CliniMACS Buffer and mesh filtered. All these steps were conducted in an automated manner directed by an optimized program (available upon request). Beyond this point, manual positive selection was conducted (CD34 MicroBeads kit; Miltenyi Biotec), generally twice, to increase CD34 purity (possibly ∼100%), as analyzed by staining cells with an FITC-conjugated anti–human lineage antibody cocktail, APC-CD34, Pacific Blue-CD45 (BioLegend), and PE/Cyanine7-CD38 (eBioscience).
5
Expansion and Xenotransplantation of Human Cord Blood CD34+ Cells
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Human umbilical cord blood-derived CD34+ cells (purchased from Lonza or Stem Cell Technologies) or FACS-purified CD34+CD38-CD90+CD49f+ cells (following staining with APC-CD34 [Biolegend; 560940], PE-CD38 [BD; 347687], FITC-CD90 [Biolegend; 328113], and APC/Cy7-CD49f [Biolegend; 313611]) were cultured in IMDM (Life Technologies) containing 0.1% HSA or 0.1% PVA (Sigma P8136, 363081, or 363146), supplemented with 1% ITSX, 1% P/S/G, 10 mM HEPES. For proliferation assays, 50 cells were seeded per well and supplemented with 10 ng/ml human SCF and 100 ng/ml human TPO (Peprotech). During the cultures, media was refreshed every three days and counted at day-7. For xenograft assays, 2×103 cells were expanded for 7-days before being injected intravenously into sub-lethally-irradiated (1.5 Gy) NOG mice. Human cell chimerism in the PB was calculated at 16-weeks post-transplantation using PE/Cy7-CD45.1 (Biolegend; 110730) and V450-hCD45 antibodies (BD; 560367).
6
Multiparameter Flow Cytometry Analysis
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Cells from PB samples collected by retroorbital bleeding or BM samples were treated with ACK lysis buffer and stained with these dye-conjugated mAbs: Pacific Orange-Ly-6G/6C (Gr-1; Life Technologies), PE-Cyanin5-CD45R/B220, Alexa Fluor 700-CD4, Alexa Fluor 700-CD8a, PE-Cyanin7-CD45.1, APC-cyanin7-CD45.2, Pacific Blue-H2Kd, FITC-H2Kk, FITC-H2Kb, PE-H2Kb, Alexa Fluor 647-H2Kq, Brilliant Violet 510 Streptavidin (BioLegend), Alexa Fluor 700 Sca-1, and APC c-Kit (eBioscience). Data were acquired by FACSAria and analyzed using FlowJo software (Tree Star). Complete blood counts were obtained with MEK-6450 Celltac-α (Nihon Kohden). For flow cytometry analysis of human cell engraftment in NOG mice, BM samples were stained with these dye-conjugated mAbs: PE-HLA-A2, Pacific Blue-human CD45, FITC-conjugated anti–human lineage antibody cocktail, APC-CD34, APC-CD4, APC-CD8, PE-Cyanin7-mouse CD45 (BioLegend), PE-CD33, FITC-CD19 (BD), and PE/Cyanine7-CD38 (eBioscience).
7
Pluripotency and NK Cell Induction Analysis
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For analysis of pluripotency markers, iPSCs were dissociated into single cells using TrypLETM Select (Gibco), blocked with 10% human AB serum, stained with Alexa Fluor 488-SSEA-3 and Alexa Fluor 647-TRA-1-60 (BioLegend, San Diego, CA, USA), and analyzed by BD FACS Canto flow cytometer (BD Biosciences) with FlowJo software (V10.4.1). For phenotypic analysis of HPC induction and NK commitment, the cells were resuspended in FACS buffer (PBS with 2% bovine serum albumin), incubated with antibody cocktail for 30 min at 4 °C in the dark, and analyzed by BD FACS Canto flow cytometer. The antibodies used in this study included FITC-CD16, FITC-Nkp46, PE-CD56, PE-Nkp44, PerCP-CD45, PerCP/Cy5.5-CD94, APC-CD34, PE/Cy7-CD43, PE/Cy7-KIR, Alexa Fluor 700-CD161, and BV605-Nkp30 (BioLegend).
8
CD34+ HSPC Colony Assay
9
Characterization of Mesenchymal Stem Cells
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The mSGSCs were characterized based on the 3 different culture media for mesenchymal, endothelial, fetal, and hematopoietic cell membrane markers of stem cells, such as CD90/Thy-1, CD73, CD146/MUC18, STRO-1, CD105, CD106, CD34, and the embryonic marker SSEA-4, as well as glandular epithelial cell markers (cytokeratins 7/8, 14, 18) by flow cytometry as described previously (Andreadis et al. 2014 (link)). For flow cytometry, the following fluorochrome conjugated mouse anti-human antibodies were used: CD90/Τhy-1-FITC, CD73-PE, CD146/MUC18-PE, STRO-1-FITC, CD105/endoglin-APC, CD106/VCAM-1-APC, CD34-APC, SSEA-4-FITC, cytokeratins 7/8-ΡΕ, cytokeratin 14-FITC, and cytokeratin 18-FITC (all BioLegend, Fell, Germany). Analysis was performed with the Guava® cytometer easyCyte 8HT benchtop flow cytometer (Merck Millipore, USA). A total of 50,000 events per sample were recorded, and data were analyzed with the software GuavaSoft 3.1.1 and Summit 5.1. In addition to determining the percentage of cells positive for each marker, the cell size and cell internal complexity (granularity) distribution profiles were analyzed by forward scatter (FSC) vs side scatter (SSC) fluorescence intensity plots, respectively.
10
Phenotypic Characterization of Isolated Cells
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Isolated cells were fixed with 2% formalin and stained with CD34-APC (Biolegend, 343607), CD90-PE (Biolegend, 328109), CD105-PE (Biolegend, 323205), CD146-APC (Biolegend, 361015), IgG2a-APC (Biolegend, 400221), or IgG1-PE (Biolegend, 400112) for 10 min at 4°C. Following the staining, the cells were washed with 1X PBS containing 0.5% BSA and 0.1% sodium azide and analysed by flow cytometry (FACS Canto II, BD Biosciences).
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