Blood samples and bone marrow aspirate for immunophenotype analysis was obtained prior to plerixafor administration on day -5 and after its administration on day -4 (prior to thiotepa administration). The cell content was phenotyped by flow cytometry using BD FACSCanto™ II flow cytometer, BD FACSDiva 6.0 software, and a red cell lysis/multi-color antibody protocol. The following monoclonal antibodies against cell surface or intracellular markers were used: Anti-CD45 APC-H7 (Clone 2D1), Anti-CD33 PE-Cy7 (Clone P67.6), Anti-sCD3 V450 (Clone UCHT1), Anti-CD7 FITC (Clone M-T701), Anti-CD5 PE-Cy7 (Clone L17F12), Anti-CD19 APC (Clone SJ25C1), Anti-CD33 APC (Clone P67.6), Anti-HLADR APC-H7 (Clone L243), Anti-CD184 (CXCR4) PE (Clone ID9), Anti-IgG 2a PE (Clone X-39; all 6from BD Biosciences, San Jose, CA); Anti-CD34 PerCP (Clone 581), Anti-cCD3 PerCP (Clone SK7; all from Biolegend, San Diego, CA); Anti-CD38 FITC (Clone T16; Beckman Coulter, Pasadena, CA) and Anti-CD133 APC (Clone AC133, Miltenyi Biotec, Cambridge, MA). Patient-specific combinations of 6 or 8 antibodies and Boolean gating scheme were used to identify the blasts for each patient and determine their CXCR4 expression. Matched isotype control was used to determine the upper limit of fluorescent background.
Anti cd33 apc
Manufactured by BD
Sourced in United States
Anti-CD33-APC is a flow cytometry reagent that binds to the CD33 antigen expressed on the surface of certain cell types. It is labeled with the fluorescent dye Allophycocyanin (APC) for detection purposes. The core function of this product is to enable the identification and analysis of CD33-positive cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45 apc h7, by BD (2 mentions)
Anti hladr apc h7, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Anti cd14 percp cy5, by BD (2 mentions)
Facsdiva 6, by BD (1 mentions)
Anti cd19 apc, by BD (1 mentions)
Anti igg2a pe, by BD (1 mentions)
Anti cd133 apc, by Miltenyi Biotec (1 mentions)
Anti cd33 pe cy7, by BD (1 mentions)
Anti cd5 pe cy7, by BD (1 mentions)
Anti cd34 percp, by BioLegend (1 mentions)
Anti cd38 fitc, by Beckman Coulter (1 mentions)
Anti cd184 cxcr4 pe, by BD (1 mentions)
Anti cd11b pe cy7, by BD (1 mentions)
Zombie nir dye, by BioLegend (1 mentions)
Anti cd15 fitc, by BD (1 mentions)
Beriglobin, by CSL Behring (1 mentions)
Anti hla dr bv421, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Live dead tm fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti cd33 apc
1
Immunophenotypic Analysis of Leukemia Cells
2
Isolation and Immunophenotyping of Human PBMCs
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Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (GE-171440-02, Merck). Separated cells were subsequently collected from the interphase, washed with isolation buffer (2.23 g/l d -glucose, 2.2 g/L sodium citrate, 0.8 g/L citric acid, 0.5% BSA in PBS), and further centrifuged at 500 g for 10 min at RT. The cell pellet was resuspended in FBS, counted, and aliquoted 1:1 in FBS with 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich). The samples were then stored in liquid nitrogen at − 160 °C until use.
Freshly thawed PBMCs (1 × 106) were washed with RPMI and stained with Zombie NIR Dye (Biolegend) for living cell identification following the manufacturer’s instructions. Then, Fc receptors were blocked with beriglobin (50 μg/ml; CSL Behring) for 10 min at 4 °C. The antibody panel for MDSC analysis was made up of anti-CD15 FITC (1.25 µl/test, HI98 clone), anti-CD14 PerCP-CyTM5.5 (0.5 µl/test, Mφ29 clone), anti-CD11b PE-Cy7 (0.5 µl/test, ICRF44 clone), anti-CD33 APC (1.25 µl/test, WM53 clone), and anti-HLA-DR BV421 (0.5 µl/test, G46-6 clone, all from BD Biosciences). PBMCs were fixed with 0.1% PFA and then were analyzed as described above for murine blood cells.
Freshly thawed PBMCs (1 × 106) were washed with RPMI and stained with Zombie NIR Dye (Biolegend) for living cell identification following the manufacturer’s instructions. Then, Fc receptors were blocked with beriglobin (50 μg/ml; CSL Behring) for 10 min at 4 °C. The antibody panel for MDSC analysis was made up of anti-CD15 FITC (1.25 µl/test, HI98 clone), anti-CD14 PerCP-CyTM5.5 (0.5 µl/test, Mφ29 clone), anti-CD11b PE-Cy7 (0.5 µl/test, ICRF44 clone), anti-CD33 APC (1.25 µl/test, WM53 clone), and anti-HLA-DR BV421 (0.5 µl/test, G46-6 clone, all from BD Biosciences). PBMCs were fixed with 0.1% PFA and then were analyzed as described above for murine blood cells.
3
Phenotypic Characterization of PBMCs
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After thawing, PBMCs (N = 74) were analysed by flow cytometry using anti-CD11b-PE, anti-CD14-PerCP, and anti-CD11c-APC (Immunotools, Friesoythe, Germany), anti-CD33-APC, and anti-HLA-DR-APCH7 (BD Biosciences, San Jose, CA, USA). Viability was assessed by flow cytometry using the LIVE/DEAD TM Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific, Vienna, Austria). MDSC were identified by combining anti-CD14, anti-HLA-DR, anti-CD33, and anti-CD11b. Data analysis was performed using FlowJo (v.10, Ashland, OR, USA). The percentage of cells and the count of cells/µL were determined.
4
MDSC Phenotyping in PBMCs
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To determine the frequency and phenotype of CD14+HLA-DR−/low (M-MDSCs) and HLA-DR−/lowCD33+CD11b+CD15+ (G-MDSCs) cells in PBMCs, the following labeled multicolor fluorescence anti-human monoclonal antibodies (mAbs) were used for surface staining: anti-HLA-DR-FITC, anti-CD33-APC, anti-CD11b-PE, anti-CD15-PERCP-Cy5.5, and anti-CD14-PERCP-Cy5.5 (BD Pharmingen, USA). The cells of each sample were incubated with these labeled multicolor fluorescence anti-human monoclonal antibodies at 4°C for 30 minutes. After surface staining, the cells were washed with 2 mL flow staining buffer (PBS plus 1% FBS) at 4°C and centrifuged at 400 g for 5 minutes. Cell pellets were diluted in 300 μL PBS after the supernatant was removed. Flow cytometry was done on a FACSCalibur. All FACS-data was analyzed by FlowJo software (TreeStar, Inc., Ashland, OR). Isotype-matched control antibodies were used as negative controls.
5
Comprehensive Characterization of Leukocyte Populations
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Quality control of LP, preDS, and DS was performed as previously described (Tischer et al., 2014b (link)). Total CD45+ leukocytes, viability, and frequencies as well as total cell numbers were determined using Trucount Absolute Counting Tubes (BD Biosciences) and the following staining reagents: anti-CD45 APC-H7 and anti-CD3 FITC mAbs and 7-AAD (all BD Biosciences). After staining, erythrocytes were lysed using Lysing Solution (Beckman Coulter) and samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the Trucount beads gate. Purity, memory phenotype and cellular composition were analyzed using combinations of the following staining reagents: anti-CD45 APC-H7, anti-CD3 FITC, anti-CD4 AF700, anti-CD8 APC, anti-CD14 BV510, anti-CD19 BV510, anti-IFN-γ PE, anti-CD45RA BV605, anti-CD62L BV421, anti-CD56 AF700, anti-CD33 APC, anti-CD19 PE-Cy7 mAbs, and 7-AAD (all BD Biosciences). After staining and lysis of erythrocytes, samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the CD45+ leukocyte gate.
6
In vitro Hematopoietic Differentiation Assay
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For in vitro differentiation assays, cells from each of the experimental conditions were incubated for 14 days in the same manner as described for the proliferative capacity analysis. 24-well-plates had been seeded with murine stromal cells (MS5) in a αMEM medium (Gibco, Thermo Fisher, Langenselbold, Germany) with 10% fetal calf serum and PS (100 ng/L). At the end of the incubation period in liquid culture, hematopoietic cells were co-cultivated on MS5 for 30 days with a complete RPMI medium (Thermo Fisher, Langenselbold, Germany, with SCF 100 ng/mL, IL-3 0.5 ng/mL, G-CSF 100 ng/mL, TPO 10 ng/mL, 10% fetal calf serum, and PS) at 37°C. At the end of the incubation period, hematopoietic cells from each well were harvested and labeled at 4°C for 20 min in the dark with anti-CD19-PE, anti-CD33-APC (BD, Biosciences, San Diego, CA, USA), and anti-CD45-FITC antibodies (IOTest, Beckman Coulter, Carlsbad, CA, USA), and analyzed with a flow cytometer.
7
Flow Cytometric Analysis of Immune Cells
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For flow cytometry a BD FACSCanto II flow cytometer and FACSDivaTM analysis were used. The following antibodies were purchased from BD Biosciences: anti-HLA-DR-PE-Cy7, anti-CD33-APC, anti-CD11b-APC-Cy7, anti-CD14-PerCP-Cy5.5, anti-CD15-PE-Cy7 and anti-ARG-1-FITC; and their corresponding isotype negative control antibodies: mouse IgM-PE-Cy7, mouse IgG1-APC, rat IgG2b APC-Cy7, mouse IgG2a-PerCP-Cy5.5, and mouse IgM-PE-Cy7.
8
Quantifying Circulating Monocyte HLA-DR and DC Subsets
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Quantifi cation of HLA-DR on circulating monocytes was performed using a standardised fl ow cytometric assay (QuantiBRITE HLA-DR, BD Biosciences, Heidelberg, Germany), as described elsewhere [17] . Enumeration of DC subsets was performed as described elsewhere [16] . In short, 150 μ L of whole blood was stained with fl ourescein isothiocyanate-conjugated antibodies against lineage (lin1) markers (mixture of anti-CD3/CD14/CD16/CD19/CD20/CD56), anti-CD123-PE, anti-HLA-DR-PerCP, and anti-CD33-APC (BD Biosciences, Heidelberg, Germany). PDC were gated as lin1-CD123 + HLA-DR + events and MDC as lin1-CD33 + HLA-DR + . Aft er treatment with FACS Lysing Solution, at least 300 events per DC population were analyzed on a FACSCalibur using CellQuest Pro (BD Biosciences, Heidelberg, Germany) software. HLA-DR expression on DCs was measured as mean fl uorescence intensity. Absolute APC population frequencies were calculated as WBC counts multiplied by the ratio of the APC population over all leukocytes. The gating strategy is given ( Figure 1 ).
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