Cd34 apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD34-APC is a lab equipment product that is used to detect and analyze CD34-positive cells. It is a fluorescent-labeled monoclonal antibody that specifically binds to the CD34 antigen on the surface of cells. The APC (Allophycocyanin) fluorochrome allows for the detection and quantification of CD34-positive cells through flow cytometry.

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Anti-CD34 APC clone 581

23 protocols using cd34 apc

Viability Assay of AML Cells

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Primary AML samples were treated with the compounds or vehicle (0.25% DMSO) for 7 days. Cells were collected and stained with mouse anti-human CD45-APC-H7 (BD Bioscience), CD34-APC and CD38-PE-Cy7 (eBiosciences) prior to Annexin V (BD Bioscience) and 7-AAD (Life Technologies) staining. Cells were then analyzed using an LSR II flow cytometer (BD Bioscience) collecting at least 2 × 10⁵ events for each sample. Data analysis was performed using FlowJo 9.3.2 software for Mac OS X (TreeStar). Annexin V and 7-AAD double negative cells were scored as viable, and the viability was represented as the percent relative to DMSO treated cells.

Borkin D., He S., Miao H., Kempinska K., Pollock J., Chase J., Purohit T., Malik B., Zhao T., Wang J., Wen B., Zong H., Jones M., Danet-Desnoyers G., Guzman M.L., Talpaz M., Bixby D.L., Sun D., Hess J.L., Muntean A.G., Maillard I., Cierpicki T, & Grembecka J. (2015). Pharmacologic inhibition of the menin-MLL interaction blocks progression of MLL leukemia in vivo. Cancer cell, 27(4), 589-602.

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Characterization of PPAR-stimulated iMSC-Derived Extracellular Vesicles

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Pan PPAR-iMSC-EVs were stained using human MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific). For analyzing the effect of pan PPAR-iMSC-EVs on hepatocyte regeneration, the hepatocytes were stained with anti-human CD90 APC-Cy7 antibody (BioLegend, San Diego, CA, USA) after pan PPAR-iMSC-EVs treatment and analyzed using the Attune NxT flow cytometer (Thermo Fisher Scientific). To confirm whether pan PPAR agonist-stimulated iMSCs express the typical cell surface markers for MSCs, pan PPAR agonist-stimulated iMSCs were stained with CD73 APC, CD105 PE, CD45 FITC, CD31 PE, and CD34 APC (eBioscience, Waltham, MA, USA) and CD90 APC-Cy7 (BioLegend) antibodies. Flow cytometric analysis was conducted using an Attune NxT flow cytometer (Thermo Fisher Scientific).

Kim J., Lee S.K., Jeong S.Y., Cho H.J., Park J., Kim T.M, & Kim S. (2021). Cargo proteins in extracellular vesicles: potential for novel therapeutics in non-alcoholic steatohepatitis. Journal of Nanobiotechnology, 19, 372.

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Isolating Hematopoietic Stem Cells

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Mononuclear cells were isolated from 5 young (24–37 years old) donors and 4 aged (64–71 years old) donors with Ficoll-Paque (GE Healthcare), then enriched using CD34 MicroBeads on a Automacs Pro separator (Miltenyi, San Diego, CA). CD34+ cells were stained with lineage antibodies conjugated to PerCP-Cy™5.5: CD2 (Becton, Dickinson and Company, clone RPA-2.10), CD3 (Becton, Dickinson and Company, clone UCHT1), CD4 (Becton, Dickinson and Company, clone RPA-T4), CD7 (Becton, Dickinson and Company, clone M-T701), CD8 (Becton, Dickinson and Company, clone RPA-T8), CD10 (Becton, Dickinson and Company, clone HI10a), CD11b (Biolegend, clone ICRF44), CD14 (Becton, Dickinson and Company, clone M5E2), CD19 (eBioscience, clone HIB19), CD20 (eBioscience, clone 2H7), CD24 (Biolegend, clone ML5), CD56 (Becton, Dickinson and Company, clone B159), CD66b (Becton, Dickinson and Company, clone G10F5), Glycophorin A (Biolegend, clone HIR2) and CD90-biotin (eBioscience, clone 5E10). To isolate HSCe, lineage stained cells were stained with: Streptavidin APC-Cy7 (Becton, Dickinson and Company), CD123-PE (eBioscience, clone 6H6), CD34-APC (eBioscience, clone 4H11), CD38-PE-Cy7 (eBioscience, clone HIT2), CD45RA-FITC (Invitrogen, clone MEM-56). Cell sorting was performed on a BD FACSAria II with a 100 μm nozzle.

Adelman E.R., Huang H.T., Roisman A., Olsson A., Colaprico A., Qin T., Lindsley R.C., Bejar R., Salomonis N., Grimes H.L, & Figueroa M.E. (2019). Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia. Cancer discovery, 9(8), 1080-1101.

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Multicolor Flow Cytometry Analysis

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Cells resuspended in PBS were stained for different FACS antibodies and subsequently incubated in dark under room temperature for 20 minutes. Then cells were washed and analyzed by FACS Fortessa. CD34-APC(Cat#17–0349)/PE-Cy7(Cat#25–0349), CD71-APC(Cat#17–0719)/ PE-Cy7(Cat#25–0719), Glycophorin A(GPA)-APC(Cat#17–9987), B220-APC(Cat#17–0452), CD19-PE(Cat#12–0199), CD3-PE-Cy7(Cat#25–0038), CD41-PE-Cy7(Cat#25–0419), CD14-PerCP-eFluor610(Cat#61–0149)/PE-Cy7 (Cat#25–0149), Annexin V-APC(Cat#88–8007), and human CD45-PE (Cat#12–9459) antibodies were purchased from eBioscience; Glycophorin A (GPA)-PE(Cat#555570), CD11b-BV421(Cat#562632)/BV605(Cat#562721), CD19-BV605(Cat#562653) were purchased from BD Biosciences.

Zhao K., Zheng W.W., Dong X.M., Yin R.H., Gao R., Li X., Liu J.F., Zhan Y.Q., Yu M., Chen H., Ge C.H., Ning H.M., Yang X.M, & Li C.Y. (2018). EDAG promotes the expansion and survival of human CD34+ cells. PLoS ONE, 13(1), e0190794.

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Enrichment and Proliferation of CD34+ Cells

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Patient samples were obtained from the Manchester Cancer Research Centre Biobank (HTA 30004) and had ethical approval from the National Research Ethics Service committee (14/LO/0489). Experimental details are available in Supplementary Methods. CD34⁺ cells were enriched using CliniMACS (Miltenyi Biotec, Bisley, UK) and colony-forming assays performed in methylcellulose complete media (R&D Systems, Abingdon, UK) supplemented with 2 U/ml erythropoietin at a density of 3000 cells per ml. To assess retention of self-renewal capacity, the resulting colonies at day 7 were replated in methylcellulose and colonies counted at 14 days. CD34⁺ cells were stained using CellTrace Violet Cell Proliferation Kit (Molecular Probes, Thermo Fisher) and then seeded at a density of 1.5 × 10⁵/ml in Iscove’s modified Dulbecco’s medium, 20% (v/v) fetal calf serum, recombinant human interleukin-3 (20 ng/ml), recombinant human stem cell factor (50 ng/ml) and Flt-3 (Fms-related tyrosine kinase 3) ligand (10 ng/ml) (PeproTech, London, UK). On day 0 and following 8 days of culture, cells were harvested and stained with CD34-APC (eBioscience, Thermo Fisher) and appropriate controls using standard protocols. Fluorescence was measured on a LSRFortessa (Becton Dickenson, Oxford, UK) flow cytometer. Results were analysed using FloJo software (Ashland, OR, USA).

Pearson S., Williamson A.J., Blance R., Somervaille T.C., Taylor S., Azadbakht N., Whetton A.D, & Pierce A. (2017). Proteomic analysis of JAK2V617F-induced changes identifies potential new combinatorial therapeutic approaches. Leukemia, 31(12), 2717-2725.

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Immunophenotyping of Mesenchymal Stromal Cells

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After digesting NP-MSCs with 0.25% trypsin (Biosharp, United States), the cells were washed and resuspended in 100 µl PBS (Sigma, United States) containing 1% FBS. Each tube contained 5 µl of the following antibodies, according to the recommendations of the International Society for Cell Therapy: CD34-APC, CD73-FTTC, CD45-PE, CD90-FTTC, CD105-PE, and HLA-DR-APC (eBioscience, United States). In each case, isotype control (eBioscience, United States) was used. After incubation with the antibody for 30 min at room temperature, the cells were washed with PBS, and the supernatant was discarded. The cells were resuspended in 200 ul of PBS (Sigma, United States) and analyzed using a flow cytometer (BD, United States). Immunophenotyping analysis was performed to identify the percentage of positive cells and the fluorescence intensity.

Wang Z., Han L., Chen H., Zhang S., Zhang S., Zhang H., Li Y., Tao H, & Li J. (2022). Sa12b Improves Biological Activity of Human Degenerative Nucleus Pulposus Mesenchymal Stem Cells in a Severe Acid Environment by Inhibiting Acid-Sensitive Ion Channels. Frontiers in Bioengineering and Biotechnology, 10, 816362.

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Flow Cytometric Analysis of Extracellular Vesicles

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Aliquots of blood plasma 16,000g and 160,000g pellets (fractions III and IV) were stained with mouse anti-human CD3-FITC, CD79a-PerCP-Cy5.5, CD41a-FITC, CD34-APC, and CD63-PE according to the manufacturer's recommendations (eBioscience, USA). Pellets were washed twice with dfPBS and analysed by FACSCanto II (Becton Dickinson, USA). Forward scatter and side scatter (FSC and SSC) PMT voltage settings were adjusted for the detection of blood vesicles/extracellular particles (60–1000 nm) using CST beads (Becton Dickinson, USA) and 60 nm polystyrene beads (Thermo Scientific, USA). PMT voltage settings for the detection of FITC, PE, PerCP-Cy5.5, and APC fluorescence were adjusted using Anti-Mouse Ig, κ/Negative Control Compensation Particles Set beads, according to manufacturer's recommendations (Becton Dickinson, USA). The following settings were used for flow cytometry analysis: FSC, 615; SSC, 310; FITC, 548; PE, 466; PerCP-Cy5.5, 505; APC, 300; threshold FSC/SSC, 200/200; compensation PE-FITC, 18; and compensation PerCP-Cy5.5/PE, 15. Gates were set according to unstained samples. Flow cytometry data were analysed with BD FACSDiva software v6.1.3 (Becton Dickinson, USA). Overlaid histograms were created using Flowing software v2.5.1 (Turku University, Finland).

Savelyeva A.V., Kuligina E.V., Bariakin D.N., Kozlov V.V., Ryabchikova E.I., Richter V.A, & Semenov D.V. (2017). Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions. BioMed Research International, 2017, 7404912.

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Comprehensive Antibody Validation for Diverse Assays

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Antibodies used for western blots (all diluted by 1:2000) include anti-Flag (M2)-HRP (Sigma, A8592), anti-H3 (CST, 9715), anti-GAPDH (CST, 2118), anti-β-Tubulin (CST, 2146), Streptavidin-HRP (CST, 3999) and anti-GFP (CST, 2956). Antibodies used for FACS (all diluted by 1:100) include c-Kit^APC (Invitrogen, 17-1172-82), c-Kit^FITC (eBioscience,11-1171-85), Cd34^APC (eBioscience, 50-0341-82), Cd34^FITC (BD, 560238), Mac1^APC (BD, 557686), Mac1^FITC (eBioscience, 11-0112-85), Gr1^FITC (eBioscience, 11-5931-85), Cd4^FITC (eBioscience,11-0042-82), Cd8a^FITC (eBioscience,11-0081-82), and Cd19^FITC (eBioscience,11-0193-82). Antibodies used in ChIP, ChIP-seq and CUT&RUN assays include anti-Flag (Sigma, F1804), anti-HA (Abcam, ab9110), anti-GFP (Abcam, ab290), anti‐H3K36me3 (Abcam, ab9050), anti-H3K27ac (Abcam, ab4729), anti‐H3K27me3 (Millipore, 07-449), anti-H4ac (Millipore, 06-866), anti-BRD4 (Bethyl, A301-985A100) and anti-Tip60 (Santa Cruz, sc-166323). 10 ug antibodies were mixed with 100 μl Dynabeads for each ChIP or ChIP-seq assay. All antibodies used in CUT&RUN assays were diluted by 1:100.

Li J., Galbo PM J.r., Gong W., Storey A.J., Tsai Y.H., Yu X., Ahn J.H., Guo Y., Mackintosh S.G., Edmondson R.D., Byrum S.D., Farrar J.E., He S., Cai L., Jin J., Tackett A.J., Zheng D, & Wang G.G. (2021). ZMYND11-MBTD1 induces leukemogenesis through hijacking NuA4/TIP60 acetyltransferase complex and a PWWP-mediated chromatin association mechanism. Nature Communications, 12, 1045.

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Isolation and Characterization of Rheumatoid Arthritis Fibroblast-Like Synoviocytes

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In the experiment, we not only used immortalized cell lines, but also applied primary cells isolated from synovial tissues (~1 mm³) of RA patients. Primary RA-FLS were isolated as described previously [16 ]. Morphology of FLS was confirmed under the light microscope (Olympus Corporation, Japan) and further characterized by flow-cytometry (Agilent, USA) with following fluorescein-labeled antibodies: CD90-APC, CD73-PE, CD14-PE and CD34-APC (eBioscience, USA). Isotype-matched control antibodies were used as methodology controls. Stained cells were then examined using flow-cytometry and data analyzed by NovoExpress software (Agilent, USA). All experiments were performed with FLS after passage four (>95% FLS purity).

Wei J., Huang X., Zhang X., Chen G., Zhang C., Zhou X., Qi J., Zhang Y, & Li X. (2023). Elevated fatty acid β-oxidation by leptin contributes to the proinflammatory characteristics of fibroblast-like synoviocytes from RA patients via LKB1-AMPK pathway. Cell Death & Disease, 14(2), 97.

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Isolation and Characterization of Stromal Vascular Fraction

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SVF was obtained from subcutaneous WAT or interscapular BAT by treatment with 2 mg/ml collagenase (Sigma) for 45 min at 37 °C. The isolated SVF was resuspended in cold Hank’s balanced salt solution (HBSS) with 2% fetal bovine serum (FBS). Cells were incubated with Ter119-PE-Cy7 (BD Pharmingen^™), CD31-PE-Cy7 (Biolegend), CD45-PE-Cy7 (Biolegend), CD34-APC (eBioscience), and Sca-1-BV421 (Biolegend) antibodies for 30 min in HBSS containing 2% FBS on ice, then washed and resuspended in solution with propidium iodide (Sigma-Aldrich). Cells were analyzed on a BD FACSAria cell sorter after selection by forward scatter (FSC) and side scatter (SSC), followed by exclusion of dead cells with propidium iodide staining, and analyzed for cell-surface markers using FlowJo software (TreeStar, Inc.). The data are shown as the percentage of Sca1⁺ and CD34 ⁺ and lineage-negative (Lin⁻) cells, as well as the number of total preadipocytes per depot.

Sakaguchi M., Fujisaka S., Cai W., Winnay J.N., Konishi M., O’Neil B., Li M., García-Martín R., Takahashi H., Hu J., Kulkarni R.N, & Kahn C.R. (2017). Adipocyte Dynamics and Reversible Metabolic Syndrome in Mice with an Inducible Adipocyte-Specific Deletion of the Insulin Receptor. Cell metabolism, 25(2), 448-462.

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