Cd33 pe

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD33-PE is a fluorescent-conjugated antibody used to detect and analyze CD33-positive cells in flow cytometry applications. It binds specifically to the CD33 antigen expressed on the surface of myeloid cells, including monocytes, granulocytes, and some progenitor cells.

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8 protocols using cd33 pe

Quantifying Myeloid Cell ROS Levels

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In vitro differentiated control or CD33 KO human myeloid cells were stained with CellROX™ Green Flow Cytometry Assay Kit and evaluated for ROS by flow cytometry according to the manufacturer’s protocol (Thermo Fisher Scientific, C10492). In brief, cells were incubated with PMA (5ng/ul) for 15 minutes at 37°C, after which the CellROX reagent was added at a final concentration of 500 nM. Cells were further incubated for 30 minutes at 37°C, then washed once with Flow Buffer and stained with CD33-PE (Thermo Fisher Scientific, 12-0339-41) and Live/Dead Fixable Aqua (Life Technologies, L34957) for 15 minutes at room temperature. Cells were then washed again with Flow Buffer, and flow cytometry was performed to quantify ROS.

Kim M.Y., Yu K.R., Kenderian S.S., Ruella M., Chen S., Shin T.H., Aljanahi A.A., Schreeder D., Klichinsky M., Shestova O., Kozlowski M.S., Cummins K.D., Shan X., Shestov M., Bagg A., Morrissette J.J., Sekhri P., Lazzarotto C.R., Calvo K.R., Kuhns D.B., Donahue R.E., Behbehani G.K., Tsai S.Q., Dunbar C.E, & Gill S. (2018). Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia. Cell, 173(6), 1439-1453.e19.

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Multi-Omics Immunology Profiling

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Analysis of samples was performed on an LSR2 (Becton Dickinson), and sorting was performed either on an ARIA (Becton Dickinson), FACS Melody (Becton Dickinson), or Fusion-instrument (Becton Dickinson). Doublets were excluded from the analysis based on FSC-H and FSC-A and dead cells were gated out using PI or Life/Death-amcyan. Labeled antibodies were obtained from BioLegend): CD3 PEcy7, CD3 APC-cy7, CD4 PerCPcy5.5, CD5-PE, CD8 amcyan, CD8 APC-Cy7, CD19 PB, CD15 PE, CD19 APC, CD20 AF-700, CD20 APC, CD27 APC-Cy7, CD33 PE, CD34 PerCPcy5.5, CD34 APC, CD41 PEcy7, CD42b APC, CD45 amcyan, CD45 PEcy7, CD56 BV421, CD69 PEcy7, CD71 APC-Cy7, CD94 PE, CD137 PE, CD235PE, HLA-DR APC-Cy7, IFN-γ PE, IL-2 PB; Thermo Fisher Scientific: CellTrace Violet Cell Proliferation Kit and Santa Cruz Biotechnology: WASp AF647.

Pille M., Avila J.M., Park S.H., Le C.Q., Xue H., Haerynck F., Saxena L., Lee C., Shpall E.J., Bao G., Vandekerckhove B, & Davis B.R. (2024). Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome. Molecular Therapy. Methods & Clinical Development, 32(1), 101208.

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Quantification of Phagocytosis in Myeloid Cells

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In vitro differentiated control or CD33 KO human myeloid cells were incubated with pHrodo green E. coli bioparticles (Thermo Fisher Scientific, P343666) for 1 hour at 37°C. Cells were then washed once with Flow Buffer (PBS with 1% FBS and 0.1% sodium azide) and stained with CD33-PE (Thermo Fisher Scientific, 12-0339-41) and Live/Dead Fixable Aqua (Life Technologies, L34957) for 15 minutes at room temperature. Cells were then washed again with Flow Buffer and acquired by flow cytometry to quantify phagocytosis.

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Immune Cell Characterization Using Flow Cytometry

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RPMI 1640, DMEM, Lipofectamine 2000, FBS, β-ME, penicillin, 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), and 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) were obtained from Invitrogen (Grand Island, NY, USA). NW-hydroxy-nor-arginine (NOHA) and L-NG-monomethyl-arginine (L-NMMA) were obtained from Cayman Chemical (Ann Arbor, MI), N-acetylcysteine (NAC) and dimethyl sulfoxide were purchased from Sigma-Aldrich (Merck, Germany). The following anti-human Abs was purchased from Thermo Fisher Scientific (Waltham, MA, USA): CD11b-FITC, CD33-PE, CD14-PE-Cy7, CD15-eFluor450, HLA-DR-PE-Cy5, CD4-PE, CD8-PE-Cy5, CD3-PE-Cy7, and their corresponding isotope controls.

Liu Y.F., Zhuang K.H., Chen B., Li P.W., Zhou X., Jiang H., Zhong L.M, & Liu F.B. (2018). Expansion and activation of monocytic-myeloid-derived suppressor cell via STAT3/arginase-I signaling in patients with ankylosing spondylitis. Arthritis Research & Therapy, 20, 168.

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Evaluation of Tamibarotene Treatment in AML Xenograft Model

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All animal experiments were performed with approval of the Baylor College of Medicine Institutional Animal Care and Use Committee. Briefly, 6- to 7-week-old female NSG-SGM3 (NSGS) mice were purchased from The Jackson Laboratory and injected by tail vein with 2 × 10⁵ primary pAML patient samples (p401, p198). Engraftment was monitored by peripheral blood with human CD45-PE (560178; BD Biosciences) and CD33-PErCP-Cy7 (561160; BD Biosciences) every 7 to 14 days. Once engraftment was confirmed (defined as >1% hCD45⁺/hCD33⁺ cells in the periphery), mice were randomized to be given either vehicle or tamibarotene treatment (n = 5 to 6 per arm). Tamibarotene was prepared as a 100× stock solution in dimethyl sulfoxide and stored at −20°C. Mice were treated with 6 mg/kg tamibarotene daily by oral gavage until moribund, at which point the mice were euthanized. Peripheral blood, liver, spleen, bilateral femurs, tibias, and fibulas were collected. Bone marrow cells were analyzed flow cytometrically by using CD45-APC-H7 (560178; BD Biosciences), CD33-PE (12-0331-82; Thermo Fisher Scientific), and CD38-FITC (555459; BD Bioscience). Tamibarotene-treated MV4;11 cells were used as positive controls for the flow experiments. Survival was plotted in GraphPad Prism by using a Kaplan-Meier analysis.

Perez M.W., Sias-Garcia O., Daramola A., Wei H., Terrell M., Rashid R., Park W.D., Duong K., Horton T.M., Li F., Cherayil N., Koren J.V., Gant V.U., Junco J.J., Curry C.V., Stevens A.M., Lin C.Y, & Yi J.S. (2021). Defining the transcriptional control of pediatric AML highlights RARA as a superenhancer-regulated druggable dependency. Blood Advances, 5(23), 4864-4876.

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Macrophage Phenotype Characterization

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Cells were detached using Cell Dissociation Solution Non-enzymatic (Sigma-Aldrich) and stained with fluorochrome-conjugated antibodies against CD163-FITC, CD11b-APC (both from BioLegend), CD206-FITC (BD BioSciences), MSR1-Alexa Fluor^® 700 (R&D Systems, MN, USA) and CD33-PE (eBioscience) at 5 and 20 days after M-CSF or M-CSF/RANKL stimulation. Cells (50 × 10³) were acquired using a Gallios Flow Cytometer (Beckman Coulter, Pasadena, CA, USA) and analyzed by FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Garcia-Gomez A., Li T., Kerick M., Català-Moll F., Comet N.R., Rodríguez-Ubreva J., de la Rica L., Branco M.R., Martín J, & Ballestar E. (2017). TET2- and TDG-mediated changes are required for the acquisition of distinct histone modifications in divergent terminal differentiation of myeloid cells. Nucleic Acids Research, 45(17), 10002-10017.

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Mechanistic Investigation of Anti-Inflammatory Compounds

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RPMI 1640, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from GE healthcare (PAA, Pasching, Austria); Anti-CD54-FITC was purchased from Biolegend (San Diego, CA, USA). Anti-CD13-FITC, CD33-PE, anti-CD15-PE, and anti-CD11b- PE were purchased from Affymetrix (eBioscience, San Diego, CA, USA). Primary antibodies for p-MEK1/2, total MEK1/2, p-P44/42 MAPK, total P44/42 MAPK, p-JNK, total JNK, p-P38, total P38, ICAM-1, p-P65, and total P65 were all obtained from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies for p-STAT1 and total STAT1 were purchased from Beckton Dickinson (BD Biosciences, San Jose, CA, USA). Anti-β-actin and horseradish peroxidase coupled secondary antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Horseradish peroxidase coupled secondary antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).
Dimethyl sulfoxide (DMSO), all-trans retinoic acid (ATRA), and celastrol were all purchased from Sigma-Aldrich (St Louis, MO, USA). ATRA and celastrol were dissolved to 100 mM in DMSO, stored at −20°C, and used within three month. ATRA and celastrol was further diluted with culture medium (for cell culture experiments) or PBS (for animal experiments) just before use.

Xu L.M., Zheng Y.J., Wang Y., Yang Y., Cao F.F., Peng B., Xu X.F., An H.Z., Zheng A.X., Zhang D.H., Uzan G, & Yu Y.Z. (2014). Celastrol Inhibits Lung Infiltration in Differential Syndrome Animal Models by Reducing TNF-α and ICAM-1 Levels while Preserving Differentiation in ATRA-Induced Acute Promyelocytic Leukemia Cells. PLoS ONE, 9(8), e105131.

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Monocytic Differentiation of HSPCs

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Briefly, 50,000 HSPCs were cultured in low-attachment 24-well plates with StemSpan (STEMCELL Technologies) supplemented with Flt-3L (50 ng/mL), SCF (200 ng/mL), IL-3 (10 ng/mL), IL-6 (50 ng/mL), and macrophage colony stimulating factor (M-CSF) (50 ng/mL) (all from PeproTech) during 21 days. Media were renewed every 2–3 days. Monocytic differentiation was determined by the increment of FSC and SSC and expression of CD33-PE and CD14-APC (all from eBioscience) by flow cytometry.

Muñoz P., Tristán-Manzano M., Sánchez-Gilabert A., Santilli G., Galy A., Thrasher A.J, & Martin F. (2020). WAS Promoter-Driven Lentiviral Vectors Mimic Closely the Lopsided WASP Expression during Megakaryocytic Differentiation. Molecular Therapy. Methods & Clinical Development, 19, 220-235.

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