Apc conjugated anti cd34

Manufactured by BD

Sourced in United States

APC-conjugated anti-CD34 is a monoclonal antibody that binds to the CD34 antigen, which is expressed on hematopoietic stem and progenitor cells. The antibody is conjugated with allophycocyanin (APC), a fluorescent dye that can be detected using flow cytometry.

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6 protocols using apc conjugated anti cd34

Surface Marker Expression Profiling

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Cells were harvested and analyzed for surface expression using the following antibodies: APC conjugated anti-CD34, PE-conjugated anti-CD71, FITC-conjugated anti-glycophorin A (BD Biosciences). 200,000 cells were collected washed and stained with the respective antibodies. All samples were analyzed within 1 hour following antibody staining using the BD LSR Fortessa Cell Analyzer in combination with BD FACSDiva and FlowJo Software.

Walker A.L, & Ofori-Acquah S. (2016). Sustained enhancement of OCTN1 transporter expression in association with hydroxyurea induced gamma-globin expression in erythroid progenitors. Experimental hematology, 45, 69-73.e2.

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Purification and Analysis of Mouse LT-HSCs and MEPs

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Mouse LT-HSCs were purified from BM cells of SclCre mutant mice 12 wk after tamoxifen injection. BM mononuclear cells were stained with an allophycocyanin (APC)-conjugated anti–c-Kit antibodies (BioLegend). c-Kit–positive cells were isolated with goat anti–APC microbeads (Miltenyi Biotec) through an LS column (Miltenyi Biotec). The c-Kit–positive cells were further stained with antibody cocktail consisting of biotinylated anti–Gr-1, Mac-1, IL-7Rα, B220, CD4, CD8α, and Ter119 monoclonal antibodies (lineage-marker cocktail), PE-conjugated anti-CD150, PE-Cy7–conjugated anti-CD48, and brilliant violet 421 (BV421)–conjugated anti–Sca-1 antibodies. Biotinylated antibodies were detected with streptavidin-APC-Cy7 (BioLegend). Mouse MEPs were also purified from spleen cells of SclCre mutant mice 12 wk after tamoxifen injection. In brief, c-Kit–positive cells were isolated using PE-conjugated anti–c-Kit antibody and anti-PE microbeads with LS column (Miltenyi Biotec). The c-Kit–positive cells were further stained with lineage-marker cocktail followed by PE-Cy7–conjugated anti-CD16/32 (BioLegend), APC-conjugated anti-CD34 (BD), streptavidin-APC-Cy7, and BV421-conjugated anti–Sca-1 antibodies. Cell sorting was performed by Influx (BD), and results were analyzed with FlowJo software (Tree Star).

Shimizu T., Kubovcakova L., Nienhold R., Zmajkovic J., Meyer S.C., Hao-Shen H., Geier F., Dirnhofer S., Guglielmelli P., Vannucchi A.M., Feenstra J.D., Kralovics R., Orkin S.H, & Skoda R.C. (2016). Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis. The Journal of Experimental Medicine, 213(8), 1479-1496.

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CD34+ cell expansion and single-cell analysis

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CD34⁺ cells from human umbilical cord blood were cultured in expansion culture medium for 7 days in the presence of TNFSF15 at 37°C with 5% CO₂. Then the cells were collected, respectively, and stained with APC‐conjugated anti‐CD34 (BD; 555824), PE.Cy7‐conjugated anti‐CD38 (BD; 560677), APC.Cy7‐conjugated anti‐CD45RA (BD; 560674), PerCP.Cy5.5‐conjugated anti‐CD90 (BD; 561557) and PE‐conjugated anti‐CD49f (BD; 555736) antibodies for 30 minutes at room temperature in dark. Then 50 CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ cells were sorted into a mixture of CellsDirect 2× Reaction Mix, 0.2× TaqMan Assay Mix (Applied Biosystems) and SuperScript III RT/ PlatinumTaq Mix (Invitrogen). Total RNA was extracted with CellsDirect One‐Step qRT‐PCR Kit (Invitrogen) according to the manufacturer's instructions. Reverse transcription and specific target amplification were performed continuously with the following parameters: 50°C for 15 minutes, 95°C for 2 minutes, 95°C for15 s for 18 cycles and 60°C for 4 minutes. Pre‐amplified cDNA was diluted with TE buffer (1:5), and PCR assay was performed. Data were analysed using BioMark Real‐Time PCR Analysis Software (Fluidigm).

Ding Y., Gao S., Shen J., Bai T., Yang M., Xu S., Gao Y., Zhang Z, & Li L. (2020). TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by activating Notch signal pathway. Journal of Cellular and Molecular Medicine, 24(19), 11146-11157.

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Quantification of Endothelial Progenitor Cells

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200 µl of whole blood per mouse was collected. Red blood cells were lysed with 1mL lysis solution, as per the manufacturer’s instructions (Qiagen, Alameda, CA). The cells were washed twice with 10mL of 1% BSA-PBS, resuspended in 1mL of 1% BSA-PBS solution, and counted. EPCs were co-labeled using APC-conjugated anti-CD34 (1 µl per 5×10⁶ cells), FITC-conjugated anti-CD133 (1.5 µl per 1×10⁶ cells), and PE-conjugated anti-Flk-1 (2 µl per 1×10⁶ cells) monoclonal antibodies (BD Biosciences, San Jose, CA). Incubations with the antibodies were performed in the dark for 20 min at 4 °C with gentle rocking. Unstained control and individual color controls were also included for gating. Cells were washed and re-suspended in 350 µL of medium containing 7-AAD viability stain for live/dead discrimination (3µL per 1×10⁶ cells, eBioscience, San Diego, CA). With the FACS Canto-II flow cytometer (BD Biosciences, San Jose, CA), single cells were gated to obtain the live CD34 positive population (Figure 5A). Within this population, cells that co-expressed CD133 and Flk-1 were counted as EPCs (Figure 5B–C). Each data point included at least 1,000,000 events. Flow data were then analyzed with FlowJo software (Tree Star Inc., Ashland, OR) by a blinded investigator.

Balaji S., Han N., Moles C., Shaaban A.F., Bollyky P.L., Crombleholme T.M, & Keswani S.G. (2015). Angiopoietin-1 Improves Endothelial Progenitor Cell-Dependent Neovascularization in Diabetic Wounds. Surgery, 158(3), 846-856.

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Flow Cytometric Characterization of EPCs

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EPCs were digested with 0.25% trypsin, washed three times with PBS, and incubated for 30 min on ice with 100 μl PBS containing the surface marker antibodies such as FITC-conjugated anti-CD133, APC-conjugated anti-CD34, PE-conjugated anti–cluster of differentiation 31 (CD31), FITC-conjugated anti-CD14, APC-conjugated anti-CD45, and anti-kinase insert domain receptor (KDR) (BD Biosciences, San Jose, CA, USA). Quantitative flow cytometry was performed using FACS Calibur (BD Biosciences), and data were analyzed using FlowJo software (BD Biosciences).

Ma W., Zhong T., Chen J., Ke X., Zuo H, & Liu Q. (2022). Exogenous H2S reverses high glucose-induced endothelial progenitor cells dysfunction via regulating autophagy. Bioengineered, 13(1), 1126-1136.

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Quantification of Endothelial Progenitor Cells

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350 µl of whole blood per mouse was collected. Red blood cells were lysed using lysis solution per manufacturer's instructions (Qiagen, Alameda, CA). The cells were washed and centrifuged twice in 10 ml of 1% BSA‐PBS and re‐suspended in 1 ml of 1% BSA‐PBS solution and counted. EPCs were co‐labeled using APC‐conjugated anti‐CD34 (1 µl per 5 × 10⁶ cells), FITC‐conjugated anti‐CD133 (1.5 µl per 1 × 10⁶ cells), and PE‐conjugated anti‐Flk‐1 (2 µl per 1 × 10⁶ cells) monoclonal antibodies (BD Biosciences, San Jose, CA) in the dark for 20 min at 4°C with gentle rocking. Unstained control and individual color controls were also included for gating. Cells were washed and re‐suspended in 350 µl of medium containing 7‐AAD viability stain for live/dead discrimination (3 µl per 1 × 10⁶ cells, eBioscience, San Diego, CA). Using the FACS Canto‐II flow cytometer (BD Biosciences, San Jose, CA), single cells were gated to obtain a live CD34‐positive population. Within this population, cells that co‐expressed CD133 and Flk‐1 were counted as EPCs. Each data point included at least 1 000 000 events. Flow data were then analyzed using the FlowJo software (Tree Star Inc., Ashland, OR) by a blinded investigator.

Short W.D., Steen E., Kaul A., Wang X., Olutoye OO I.I., Vangapandu H.V., Templeman N., Blum A.J., Moles C.M., Narmoneva D.A., Crombleholme T.M., Butte M.J., Bollyky P.L., Keswani S.G, & Balaji S. (2022). IL‐10 promotes endothelial progenitor cell infiltration and wound healing via STAT3. The FASEB Journal, 36(7), e22298.

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