Sixteen to18 weeks after transplantation, samples of peripheral blood, bone marrow, and spleen were harvested from recipient mice. Samples were treated with ammonium chloride to eliminate mature erythrocytes. Non-specific antibody binding was blocked (10% vol/vol, TruStain FcX, BioLegend), cells were stained (30 min, 4 °C, dark), and analyzed by setting nucleated cell scatter gates using a BD FACSAria II flow cytometer or BD FACSCanto II analyzer (BD Biosciences). Cells were analyzed based on monoclonal anti-human HLA-ABC APC-Cy7 (W6/32, BioLegend), anti-mouse CD45.1 PE-Cy7 (A20, eBioScience, San Diego, CA, USA), CD19 APC (HIB19, BD511 Biosciences), CD33 PE (WM53, BD Biosciences), anti-mouse mTer119 PE-Cy5 (TER-119, BD Biosciences), and CD3 PerCP/Cy5.5 (HiT3A, BioLegend) antibodies, and Propidium Iodide to detect dead cells. Human engraftment was defined as HLA-ABC+/HCD45+ cells. See Supplementary Methods for a complete Antibody list.
Cd33 pe wm53
Manufactured by BD
Sourced in United States
The CD33 PE (WM53) is a lab equipment product that serves as a fluorescent-labeled antibody. It is used for the detection and analysis of CD33-expressing cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti human hla abc apc cy7 w6 32, by BioLegend (3 mentions)
Anti mouse cd45.1 pe cy7 a20, by Thermo Fisher Scientific (3 mentions)
Trustain fcx, by BioLegend (3 mentions)
Anti mouse mter119 pe cy5 ter 119, by BD (2 mentions)
Facsaria 2 flow cytometer, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cd3 percp cy5.5 hit3a, by BioLegend (1 mentions)
Facscanto 2 analyzer, by BD (1 mentions)
Cd3 sk7, by BD (1 mentions)
Fortessa lsr, by BD (1 mentions)
Cd45 v500 hi30, by BD (1 mentions)
Cd56 ncam16.2, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Cd19 bv605 sj25c1, by BD (1 mentions)
Anti mouse igk and negative beads, by BD (1 mentions)
5 protocols using cd33 pe wm53
1
Multiparametric Flow Cytometry Analysis of Hematopoietic Engraftment
2
Multiparametric Flow Cytometry Analysis of Human Hematopoietic Engraftment in Mice
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Fifteen to 20 weeks after transplantation, mice were euthanized, and peripheral blood, BM, and spleen were collected. After several washes and treatment with ammonium chloride to remove erythrocytes, cells were treated with 10% vol/vol TruStain FcX (BioLegend, San Diego, CA, USA) to block non-specific antibody binding. Cells were stained in the dark for 30 min on ice using the following antibodies: anti-human HLA-ABC APC-Cy7 (W6/32, BioLegend), anti-mouse CD45.1 PE-Cy7 (A20, eBioScience, San Diego, CA, USA), anti-human CD45 PB (2D1, Biolegend), CD19 APC (HIB19, BD511 Biosciences), CD33 PE (WM53, BD Biosciences), anti-mouse/human TER-r119 PE-Cy5 (TER-119, BD Biosciences), and CD3 BV650 (HiT3A, BioLegend). After staining, cells were washed and resuspended in MACS buffer containing propidium iodide to detect dead cells. Samples were analyzed in a BD FACSAria II flow cytometer, and human engraftment was given by double-positive human HLA-ABC+/human CD45+ cells.
3
Evaluating Human Engraftment in Mice
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12 weeks after transplantation, mice were sacrificed, mouse bone marrow (BM) was harvested from the transplanted femur by flushing. Non-specific antibody binding was blocked (10% vol/vol, TruStain FcX, BioLegend) and cells were stained (30 min, 4°C, dark) with monoclonal anti-human HLA-ABC APC-Cy7 (W6/32, BioLegend), anti-mouse CD45.1 PE-Cy7 (A20, eBioScience, San Diego, CA, USA), CD19 APC (HIB19, BD511 Biosciences), CD33 PE (WM53, BD Biosciences), and anti-mouse mTer119 PE-Cy5 (TER-119, BD Biosciences) antibodies, and Propidium Iodide to detect dead cells. Human engraftment was defined as HLA-ABC+ cells.
4
Flow Cytometry Phenotyping of AML and Healthy PBMCs
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AML and healthy PBMC samples were phenotyped with CD45-V500 (HI30), CD34-PE-CY7 (581), CD38-V450 (HB7) and CD33-PE (WM53), CD90-AF700 (5E10), CD45RA-APC-H7 (SH9), CD11c-APC-AF700 (B-Ly6) and HLA-DR-APC-H7 (L243) mAbs from BD Biosciences or BioLegend. The lineage (Lin) cocktail consisted of CD235a (GA-R2), CD14 (MφP9), CD20 (2H7), CD19 (HIB19), CD56 (NCAM16.2) and CD3 (SK7; BD Biosciences). The mouse anti-human CD302 IgG1mAb antibody (MMRI-20) was used unlabelled or as a PE conjugate [11 (link)]. The mAb CMRF-81, specific for tetanus toxoid, was used as the mouse IgG1 isotype control [14 (link)]. DAPI (3μM; ThermoFisher) staining was used to exclude dead cells. Data were collected on Accuri C6, Canto, Fortessa LSR or Influx flow cytometers (BD Biosciences) and analysed with FlowJo 10 software (Treestar). The gating strategy for identifying BM/CB HSC and MPP are shown in panel A of S1 Fig . Binding was displayed as a geometric mean fluorescence intensity (geoMFI) ratio which was calculated by the formula: geoMFI test antibody/geoMFI isotype control. A ratio of ≥3 was considered positive. T-distributed stochastic neighbour embedding (t-SNE) visualisation was performed on FlowJo 10.
5
Comprehensive Immune Cell Analysis in Mice
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