Cd10 pe cy7

Manufactured by BD

Sourced in United States, United Kingdom

The CD10-PE-Cy7 is a fluorochrome-conjugated antibody that detects the CD10 cell surface antigen. CD10 is a cell surface metalloprotease expressed on various cell types, including B lymphocytes, granulocytes, and some malignant cells. The PE-Cy7 fluorochrome allows for the detection and analysis of CD10-positive cells using flow cytometry.

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CD10 PE (7 citations) Anti-CD10-PE-Cy7 (2 citations)

16 protocols using cd10 pe cy7

Comprehensive Neutrophil Immunophenotyping

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Neutrophils were stained for 20 min at RT with an antibody panel containing CD66b-FITC 1:20, CD10-PE-Cy7 1:20, CD11b-AlexaFluor 700 1:66, CD16-APC-H7 1:66, CD14-BrilliantViolet 421 1:66, Siglec8-BrilliantViolet 510 1:66 (all from BD) and CD62L-APC 1:66 (Biolegend). Neutrophils were washed and analyzed on a Cytoflex flow cytometer (Beckman Coulter).

Arve-Butler S., Mossberg A., Schmidt T., Welinder C., Yan H., Berthold E., Król P, & Kahn R. (2022). Neutrophils Lose the Capacity to Suppress T Cell Proliferation Upon Migration Towards Inflamed Joints in Juvenile Idiopathic Arthritis. Frontiers in Immunology, 12, 795260.

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Characterization of PBMC Immune Profiles

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PBMC from the WIHS repository were thawed and incubated for 48 hours and during the final 5 hours of incubation the cultures were supplemented with Brefeldin A (1:100, BD), PMA (50ng/ml, Invivogen) and Ionomycin (1ug/ml, Invivogen). The cells were washed, stained for viable cells (LIVE/DEAD Aqua Fixable Dead Cell Stain Kit, Invitrogen), surface stained, fixed/permeabilized (Fix/Perm Kit BD Biosciences) and stained for intracellular AID-PerCP (IC39101C, RnD System). The following antibodies were used for immunophenotyping of PBMC: CD19-ECD (IM2708U, Beckman Coulter), CD86-AF-700, CD10-PE-Cy7, CD4-Pacific Blue, CD127-APC-H7, CD71-FITC, CD38-PE, CD25-PE, CD24-PercpCy5.5 (all antibodies were purchased from BD Biosciences except when stated otherwise). All samples were acquired on an LRSII (BD, Bioscience) flow cytometer and the data were analyzed using FlowJo software (Tree Star Inc).

SIEWE B., PHAM J.T., COHEN M., HESSOL N.A., LEVINE A., MARTINEZ-MAZA O, & LANDAY A. (2015). Dysregulated B-cell TLR2 expression and Elevated Regulatory B cell frequency precede the diagnosis of AIDS-related non-Hodgkin lymphoma. AIDS (London, England), 29(13), 1659-1664.

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Flow Cytometry of Immune Cell Subsets

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The following fluorochrome conjugated antihuman monoclonal antibodies (MoAbs) were used for flow cytometry studies: ICOSBV421, ICOSAlexa488, CD40LBV605, CD69BV650, HLA-DRFITC, CD38APCCy7, and TNF-αAPCCy7 from BioLegend (San Diego, CA); CD3BUV395, CD4PerCPCy5.5, CD8Alexa-Fluor700, CCR7PECF594, IL-2BV711, CXCR5Alexa647, IFNγPE-Cy7, PD1BV650, CD45ROAPCH7, CD21PECy5, CD27PerCPCy5.5, IgDFITC, CD10PECy7, and CD20Alexa700 from BD Bioscience (San Jose, CA); IL-21PE, CD27PECy5, and IL-17Alexa488 from e-Biosciences (San Diego, CA); and CD45ROPE-TexasRed from Beckman Coulter (Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit and CellTrace Violet Cell Proliferation Kit were from ThermoFisher (Boston, MA). Recombinant human IL-21 (Cat #8879-IL-010) from R&D systems, purified antihuman IL-2 (Cat #3440-ON-500) and antihuman-IL-21 (Cat #MT216G/21.3m) from Mabtech, and antihumanTNF-α (Cat #502922) from BioLegend were used. Samples were acquired on a BD LSRFortessa (BD Biosciences, CA) flow cytometer and analyzed by FlowJo V10 (TreeStar, Inc).

Pallikkuth S., de Armas L.R., Rinaldi S., George V.K., Pan L., Arheart K.L., Pahwa R, & Pahwa S. (2019). Dysfunctional peripheral T follicular helper cells dominate in people with impaired influenza vaccine responses: Results from the FLORAH study. PLoS Biology, 17(5), e3000257.

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Phosphoflow Analysis of Leukemic Signaling

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As previously setup and validated in our lab,¹⁵ (link) the phosphoflow test was used to evaluate the levels of phosphorylation of PDPK1, Akt (Ser473), Akt (Thr308), S6 (S235/S236), 4EBP1 (Thr37/46), in leukemic cells (CD10+) derived from mice treated with vehicle or Nintedanib mice. After 1·5h serum starvation in X-VIVO at 37°C leukemic cells were then fixed for 10 minutes at room temperature by 1·5% PFA/mL and then permeabilized with 90% cold methanol, kept on ice for 30 minutes then washed with Staining Buffer. Further, cells were dark stained for 30 minutes at room temperature with antibodies directed to PDPK1 (Anti-PDPK1 pS241 #560092 RRID: AB_1645523 BD Biosciences), pAkt (Ser473) (Phospho-Akt Ser473 #4075 Cell Signaling), pAkt (Thr308) (Anti-Akt pT308 #558275 RRID: AB_2225329 BD Biosciences), pS6 (Anti-S6 pS235/pS236 #560435 RRID: AB_2869348 BD Biosciences) and 4pEBP1 (Phospho-4E-BP1 Thr37/46 #2846 Cell Signaling). Cells were also stained for CD10 (CD10 PE-Cy™7 #565282 RRID: AB_2739153 BD Biosciences), in order to clearly detect B leukemic cells.

Fazio G., Bresolin S., Silvestri D., Quadri M., Saitta C., Vendramini E., Buldini B., Palmi C., Bardini M., Grioni A., Rigamonti S., Galbiati M., Mecca S., Savino A.M., Peloso A., Tu J.W., Bhatia S., Borkhardt A., Micalizzi C., Lo Nigro L., Locatelli F., Conter V., Rizzari C., Valsecchi M.G., te Kronnie G., Biondi A, & Cazzaniga G. (2022). PAX5 fusion genes are frequent in poor risk childhood acute lymphoblastic leukaemia and can be targeted with BIBF1120. eBioMedicine, 83, 104224.

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Characterization of CD34+ Subpopulations

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The subpopulations present in the CD34⁺fraction were analyzed by flow cytometry using following monoclonal antibodies: CD34-PE (Miltenyi), CD38-APC (BD Pharmingen), CD10-PE-Cy™7 (BD Pharmingen), CD45Ra-APC-A750 (Beckman Coulter), CD90-FITC (Miltenyi), Propidium Iodide (BioLegend). Cells were co-stained with all antibodies for 15min and washed with PBS. For flow cytrometry, CytoFlex Flow Cytometer (Beckman Coulter) was used. The gating strategy is shown in Supplementary Fig. S3.

Becker F., Ouzin M., Liedtke S., Raba K, & Kogler G. (2023). DNA Damage Response After Treatment of Cycling and Quiescent Cord Blood Hematopoietic Stem Cells With Distinct Genotoxic Noxae. Stem Cells, 42(2), 158-171.

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Multi-receptor Immunophenotyping of Epithelial Cells

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For detection of EpCAM, TLR4, TLR7, NEP and NK1R, epithelial cells and biopsies were stained with the following antibodies: EpCAM‐fluoroscein‐isothiocyanate (347197; BD Bioscience), TLR4‐AlexaFluor700 (FAB6248N; RnD Systems), TLR7‐phycoerythrin (IC5875P; RnD Systems), CD10‐PE‐Cy7 (565282; BD Bioscience) and NK1R‐allophycocyanin (FAB66871A; RnD Systems). Cells were stained with aqua LIVE/DEAD® Fixable dead stain (Thermo Fisher Scientific) to detect dead cells; Fc Block (BD) was used to block nonspecific binding. Fluorescence minus one controls were used to assess background fluorescence levels. The IntraPrep Permeabilisation Reagent kit (Immmunotech; Beckman Coulter) was used to detect intracellular TLRs. Cells were analysed on an LSR Fortessa Flow Cytomter (BD Biosciences). Events in the range of 5000–30,000 were recorded. Data were analysed with FlowJo Analysis Software (TreeStar).

Larsson O., Sunnergren O., Bachert C., Kumlien Georén S, & Cardell L.O. (2021). The SP‐TLR axis, which locally primes the nasal mucosa, is impeded in patients with allergic rhinitis. Clinical and Translational Allergy, 11(1), e12009.

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Multicolor Flow Cytometry for MRD Detection

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Multicolor flow cytometry analysis of BM was performed at the day of diagnosis to assess leukemic-associated immunophenotype (LAIP) of blasts cells. A following antibodies were used for cell immunophenotyping: CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, Waltham, MA, USA), CD58 (Beckman Coulter, Brea, CA, USA). On the day 15th and 33rd BM was analyzed using the same panel of antibodies to determine MRD. To the appropriate amount of BM (10⁶ cells) antibodies listed above were added, samples were incubated for 20 min at room temperature in darkness. Erythrocytes were lysed for 10 min at room temperature in darkness with lysing solution (BD FACS Lysing Solution, Becton Dickinson Biosciences, San Jose, CA, USA), washed twice in PBS, and finally resuspended in 200 μL of PBS. For distinguishing nucleated cells, samples were stained with Syto^®41 (Thermo Fisher Scientific, Waltham, MA, USA). FACS analysis was done using FACSCanto or FACSCanto10 with FACSDiva Sorfware v. 8.1 (Becton Dickinson Biosciences, San Jose, CA, USA).

Bukowska-Strakova K., Włodek J., Pitera E., Kozakowska M., Konturek-Cieśla A., Cieśla M., Gońka M., Nowak W., Wieczorek A., Pawińska-Wąsikowska K., Józkowicz A, & Siedlar M. (2021). Role of HMOX1 Promoter Genetic Variants in Chemoresistance and Chemotherapy Induced Neutropenia in Children with Acute Lymphoblastic Leukemia. International Journal of Molecular Sciences, 22(3), 988.

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Intracellular HO-1 Expression in BM

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HO-1 expression was assessed in BM of 11 patients upon diagnosis. Then in patients in whom, after routine MRD staining, there was a sufficient material for additional staining, an intracellular HO-1 staining was performed (6 children at day 15, 3 children at day 33). In this purpose 10⁶ of bone marrow cells were stained with CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, San Jose, CA, USA), lysed, fixed, and then permeabilized using a BD Intrasure Kit, according to the manufacturer’s instructions. After permeabilization step, cells were incubated with primary antibodies recognizing HO-1 (rabbit polyclonal, SPA 896, Enzo, Warszawa, Poland), washed twice, and stained with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Life technologies, HITACHI, Tokyo, Japan). After two washing steps, samples were stained with Syto^®41 (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using FACSCanto10 with FACSDiva Sorfware v 8.0.1 (Becton Dickinson Biosciences, San Jose, CA, USA).

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Flow MRD Profiling for Acute Lymphoblastic Leukemia

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Flow MRD was performed according to the UK standardized method (13 (link)). Briefly, samples were collected into Acid Citrate Dextrose and with red cells lysed using a standard ammonium chloride procedure. Biomarker labeling was done using fluorescent-conjugated antibodies such as CD45-APCH7, CD10-PECy7, CD19-APC, CD34-PerCP (all from BD Biosciences, Plymouth, UK) and nuclear staining by Syto41 (Molecular probes, Loughborough, UK). Flow cytometric measurement was done on a BD FACs Canto II equipped with 488-nm (blue), 633-nm (red), and 405-nm (violet) lasers, with a target number of 50,000 cells for diagnostic samples and >300,000 for “on-treatment” samples. For analysis, an expert-guided sequential manual gating strategy was applied: Lymphoid cells were first gated on forward and side scatters, followed by CD19⁺, low side scatter cells, then refined inspection on CD19⁺CD34⁻ and CD19⁺/CD34⁺ double positive cells. Finally, the expression of CD10 along with CD45 was assessed in the CD19⁺CD34⁺ and CD34⁻CD19⁺ populations to discriminate ALL cells from normal B cells. Samples were considered positive if the number of leukemic cells identified was equal to or greater than 0.01% (i.e. the percentage of leukemic cells over the total number of nucleated cells), providing at least 50 clustered events were apparent.

Doan M., Case M., Masic D., Hennig H., McQuin C., Caicedo J., Singh S., Goodman A., Wolkenhauer O., Summers H.D., Jamieson D., van Delft F.W., Filby A., Carpenter A.E., Rees P, & Irving J. (2020). Label‐Free Leukemia Monitoring by Computer Vision. Cytometry, 97(4), 407-414.

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Multiparametric Flow Cytometry Analysis

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Cells were harvested and stained with antibodies at 4°C for 30 minutes, by washing with 1 mL flow cytometry staining buffer, fixed with 1% formalin solution at 4°C for 30 minutes, and resuspended with staining buffer (PBS, 0.5% BSA, and 2 mM EDTA) before analysis. For flow cytometry analysis of ATO cell harvests, a Fc receptor blocking solution, Human TruStain FcX and a fixable viability dye, zombie yellow (BioLegend, San Diego, California) were added. Antibodies (BD Biosciences, San Jose, California) used for cell surface staining were: CD34-APC (581), CD38-PE (HIT2), CD45RA-PE-CF594 (HI100), CD90-BV421 (5E10), CD10-PE-Cy7 (HI10a), CD7-APC-H7 (M-T701), CD5-BV421 (UCHT2), CD1a-FITC (HI149), CD3-FITC or APC (UCHT1), CD4-PE-CF594 (RPA-T4), CD8-PerCP-Cy5.5 (RPA-T8), TCRαβ-FITC (IP26), TCRγδ-BV421 (B1), CD19-PE (HIB19), CD33-PE-Cy7 (WM53), CD56-FITC (B159). Samples analysis was on an LSR4 flow cytometer (BD Biosciences), and data analyzed using the FlowJo software (Tree Star, Washington).

Shin D.Y., Huang X., Gil C.H., Aljoufi A., Ropa J, & Broxmeyer H.E. (2020). Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells. Stem cells (Dayton, Ohio), 38(11), 1454-1466.

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