Apc cy7 conjugated anti cd45

Manufactured by BD

APC-Cy7-conjugated anti-CD45 is a fluorescent-labeled antibody that targets the CD45 antigen. CD45 is a receptor-linked protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells. The APC-Cy7 conjugation allows for multicolor flow cytometric analysis.

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Lab products found in correlation

Fcs express version 3, by De Novo Software (2 mentions) Fitc conjugated anti cd90, by BD (2 mentions) Fix perm kit, by Thermo Fisher Scientific (2 mentions) Percp cy5.5 conjugated anti cd105, by BD (2 mentions) Pe conjugated anti cd34, by Miltenyi Biotec (2 mentions) Percp cy 5.5 conjugated anti nanog, by BD (2 mentions) Facsaria 3, by BD (2 mentions) Pe conjugated anti cd11b, by BD (2 mentions) Apc conjugated anti ly6g, by BD (2 mentions) Facscanto 2 flow cytometer, by BD (2 mentions) Accuri c6, by BD (1 mentions) 7 aad, by Thermo Fisher Scientific (1 mentions) 7 aminoactinomycin d 7 aad, by Merck Group (1 mentions) Software, by FlowJo (1 mentions) Collagenase, by Merck Group (1 mentions) Pe conjugated anti cd69, by BD (1 mentions) Fitc conjugated anti cd4, by BD (1 mentions) Apc conjugated anti cd8, by BD (1 mentions) Pe cy7 conjugated anti cd3, by BD (1 mentions) 70 μm cell strainer, by BD (1 mentions)

9 protocols using apc cy7 conjugated anti cd45

Characterization of hDPSCs by Flow Cytometry

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Flow cytometry analyses were performed on hDPSCs at first passage of culture. Cells were incubated with FITC-conjugated anti-CD90, PerCP-Cy5.5-conjugated anti-CD105, APC-Cy7-conjugated anti-CD45 (all purchased from BD Pharmingen), and PE-conjugated anti-CD34 (Miltenyi Biotech) for phenotypic characterization, and FITC-conjugated anti-bone sialo-protein (BSP) (Biorbyt), anti-CFS-conjugated anti-osteopontin (OPN) (R&D Systems) and PerCP-Cy 5.5-conjugated anti-NANOG (BD Pharmingen) for evaluation of osteogenic differentiation and mesenchymal stemness. As negative controls, cells were stained with an isotype control antibody. After incubation with the antibody, cells were re-suspended in PBS and analysed using an FACS ARIA III or BD Accuri C6 (BD Biosciences). Human DPSCs were then sorted for CD34- and CD90-positive markers. The purity of sorting was approximately 90%.
For intracellular staining of osteocalcin (OC), OPN and NANOG, cells were processed using a Fix & Perm Kit (Invitrogen) following the manufacturer's guidelines. All data were analysed using FCS express version 3 (De Novo Software).

Paino F., La Noce M., Giuliani A., De Rosa A., Mazzoni S., Laino L., Amler E., Papaccio G., Desiderio V, & Tirino V. (2017). Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering. Clinical Science (London, England : 1979), 131(8), 699-713.

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Evaluating Hematopoietic Stem Cell Markers

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Flow cytometry analyses were performed on hDPSCs cultured in medium supplemented with C-FBS or NZ-FBS at first passage of culture. Cells were incubated with FITC-conjugated anti-CD90, PerCP-Cy5.5-conjugated anti-CD105, APC-Cy7-conjugated anti-CD45 (all purchased from BD Pharmingen, San Diego, CA), and PE-conjugated anti-CD34 (Miltenyi Biotech, Calderara di Reno, Bologna, Italy) for phenotypic characterization; and anti-Bone sialoprotein (BSP) (Abcam, Cambridge, UK), anti-CFS-conjugated anti-Osteopontin (OPN), PE-conjugated anti-Osteocalcin (OC) (both from R&D Systems, Minneapolis, MN) and PerCP-Cy 5.5-conjugated anti-Nanog (BD Pharmingen, Milan, Italy) to evaluate osteogenic differentiation and mesenchymal stemness. hDPSCs were sorted by CD34 expression. The purity of sorting was 90%. As negative controls, cells were stained with an isotype control antibody. After incubation with the antibody, cells were resuspended in PBS and analyzed with a FACS ARIA III (BD Biosciences, San Jose, CA). For intracellular staining of Osteocalcin, Osteopontin and Nanog, cells were processed using Fix & Perm Kit (Invitrogen, Milan, Italy) following the manufacturer's guidelines. All data were analyzed using FCS express version 3 (De Novo Software, Glendale, CA).

Spina A., Montella R., Liccardo D., De Rosa A., Laino L., Mitsiadis T.A, & La Noce M. (2016). NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression. Frontiers in Physiology, 7, 354.

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Detecting CD45 and CD31 in Blood

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For detection of CD45 and PECAM1 (CD31), peripheral blood samples were collected, incubated with red blood lysis buffer and resulting cells were stained with allophycocyanin (APC)-cy7-conjugated anti-CD45 (BD Biosciences) and phycoerythrin (PE)-cy7-conjugated anti-CD31 (Invitrogen). Dead cells were detected by 7-AAD (Invitrogen) staining.

Ukah T.K., Cattin-Roy A.N., Davis G.E, & Zaghouani H. (2021). Formation of pancreatic β-cells from precursor cells contributes to the reversal of established type 1 diabetes. Cellular immunology, 364, 104360.

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Cell Cycle Analysis of KG1a Cells

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Detailed cell cycle analysis of KG1a cells in each condition was performed by quantifying G0, G1, S, G2, and M phases as previously reported [28 (link)]. For quantification, 10⁶ cells were permeabilized with 1mL of ice cold ethanol (1 h, 4 °C), washed twice with PBS, 1% FBS, 0.25% Triton X-100 (PFT), and stained in 200 µL of PBS-FBS-TritonX100 (PFT) for 30 min at RT in the dark with 1 µg of 7-aminoactinomycin D (7-AAD, Sigma-Aldrich, St. Louis, MO, USA), 5 µL of Alexa Fluor^®488-conjugated anti-human Ki67 mAb (B56) (BD Biosciences), and 2 µL of Alexa Fluor^®488-conjugated anti-phospho(ser10)-histone H3 polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA), respectively. After two washes with PFT, cells were stained with 10 µL of APC-Cy7-conjugated anti-CD45 (BD Biosciences) for 15 min at 4 °C. Cells were then washed twice with PBS and acquired on a FACS Canto II. Data analysis was performed with FlowJo software (v10.5.3, FlowJo, Ashland, OR, USA).

Vignon C., Debeissat C., Bourgeais J., Gallay N., Kouzi F., Anginot A., Picou F., Guardiola P., Ducrocq E., Foucault A., Ravalet N., Le Nail L.R., Domenech J., Béné M.C., Le Bousse-Kerdilès M.C., Gyan E, & Herault O. (2020). Involvement of GPx-3 in the Reciprocal Control of Redox Metabolism in the Leukemic Niche. International Journal of Molecular Sciences, 21(22), 8584.

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Activated T Cell Profiling Post-CS Exposure

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Immediately after CS exposure and lung removal, lung tissues were digested with collagenase and DNase (Sigma, St. Louis, MO, USA) to prepare single-cell suspensions by passing the dissociated tissue through a 70-μm cell strainer (BD Falcon). The suspensions of cells were stained in PBS containing 2% FBS with the following antibodies: FITC-conjugated anti-CD4, PE-Cy7-conjugated anti-CD3, APC-Cy7-conjugated anti-CD45, PE-conjugated anti-CD69, and APC-conjugated anti-CD8 antibodies (BD Bioscience). The cells were gated on FSC (forward scatter) and SSC (side scatter). The cells were then gated on SSC versus CD45 followed by SSC versus CD3 in order to gate on all T cells. The cells were further gated to obtain the percentage of CD4+ T and CD8+ T cells. Stained cells were examined on a FACS Canto flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The percentages of activated CD4+ T cells and activated CD8+ T cells were determined based on double positive cells (CD69+ CD4+ or CD69+ CD8+ cells) among total CD4+ or CD8+ T cells.

Lao T., Glass K., Qiu W., Polverino F., Gupta K., Morrow J., Mancini J.D., Vuong L., Perrella M.A., Hersh C.P., Owen C.A., Quackenbush J., Yuan G.C., Silverman E.K, & Zhou X. (2015). Haploinsufficiency of Hedgehog interacting protein causes increased emphysema induced by cigarette smoke through network rewiring. Genome Medicine, 7(1), 12.

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Renal Single Cell Analysis by Flow

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UUO (n = 6 mice) and sham (n = 6 mice) surgeries were performed, and renal single cells were isolated and analyzed by flow cytometry. Briefly, renal tissue was minced and placed into a cocktail of 0.25 mg/mL Liberase Blendzyme 3 (Roche Applied Science), 20 U/mL DNase I (Sigma-Aldrich) and shaken at 37 °C for 20 min. Subsequently, cells were passed through 40 μm nylon mesh and centrifuged (10 min, 200 g, 4 °C). Cells were stained and analyzed using flow cytometry. The following dyes and antibodies were used: APC-Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD3 (both from BD Pharmingen), PerCP/Cy5.5-labeled anti-CD31, PE/Cy7-labeled anti-F4/80 (both from Biolegend), and Cy3-conjugated anti-α-SMA (Sigma). Stained single cells were resuspended in a staining buffer and immediately analyzed with a Becton Dickinson LSRII flow cytometer (BD Biosciences).

Wu Y., Liang M., Huang F., Cheng O.H., Xiao X., Lee T.H., Truong L, & Cheng J. (2023). Notch Blockade Specifically in Bone Marrow-Derived FSP-1-Positive Cells Ameliorates Renal Fibrosis. Cells, 12(2), 214.

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Multicolor Flow Cytometry Panel

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The cells were stained with allophycocyanin (APC)‐Cy7‐conjugated anti‐CD45 (BD Pharmingen), phycoerythrin (PE)‐conjugated anti‐CD11b (BD Pharmingen), APC‐conjugated anti‐Ly6G (BD Pharmingen), BV421‐conjugated anti‐lymphocyte antigen 6 complex locus C (Ly6C) (BD Horizon), FITC Rat Anti‐Mouse CD19 (Biolegend), BV421 Rat Anti‐Mouse CD3 (BD Pharmingen), PE Rat Anti‐Mouse CD4 (Biolegend) and APC Rat Anti‐Mouse CD8a (BD Pharmingen). Dead cells and debris were gated out using forward light scatter, side light scatter, and 7‐aminoactinomycin D (BD Biosciences, San Jose, CA). The fluorescence‐activated cell sorting analysis was performed on a FACS Canto II flow cytometer and analyzed using FlowJo software (BD Biosciences).

Yan H., Kawano T., Kanki H., Nishiyama K., Shimamura M., Mochizuki H, & Sasaki T. (2023). Role of Polymorphonuclear Myeloid‐Derived Suppressor Cells and Neutrophils in Ischemic Stroke. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 12(6), e028125.

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Characterizing Immune Cell Profiles in EAE Mouse Models

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Single-cell suspensions of brains and spinal cords isolated from WT and Trim14^−/− EAE mouse models were washed in PBS with 10% FBS. Cells were stained with the following fluorescence-labeled antibodies: APC-Cy7–conjugated anti-CD45 (BD, B557659), PE-ef610–conjugated CD4 (eBioscience, 61004382), BV785-conjugated anti-CD8 (eBioscience, 60-5961), BV510-conjugated anti–IL-17A (BD, 563295) and PE-conjugated anti–IL-21 (R&D, IC594P-100). Subsequently, cell suspensions were subjected to flow cytometry for analysis. Statistical analysis was performed by Flowjo_v10.

Liu D., Zhao Z., She Y., Zhang L., Chen X., Ma L, & Cui J. (2022). TRIM14 inhibits OPTN-mediated autophagic degradation of KDM4D to epigenetically regulate inflammation. Proceedings of the National Academy of Sciences of the United States of America, 119(7), e2113454119.

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Multicolor Flow Cytometry Analysis

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The cells were stained with APC-Cy7-conjugated anti-CD45 (BD Pharmingen), PE-conjugated anti-CD11b (BD Pharmingen), APC-conjugated anti-Ly6G (BD Pharmingen), and BV421-conjugated anti-Ly6C (BD Horizon). Dead cells and debris were gated out using forward light scatter, side light scatter, and 7-AAD (BD Biosciences, San Jose, CA). FACS analysis was performed on a FACSCanto II flow cytometer and analyzed using FACS Diva software (BD Biosciences). Cell sorting was performed using FACSAria II (BD Biosciences).

Kawano T., Shimamura M., Nakagami H., Kanki H., Sasaki T, & Mochizuki H. (2019). Temporal and spatial profile of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in ischemic stroke in mice. PLoS ONE, 14(5), e0215482.

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