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Cd271 pe

Manufactured by Miltenyi Biotec
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CD271-PE is a fluorescent-labeled antibody used for the detection and identification of cells expressing the CD271 antigen, also known as the low-affinity nerve growth factor receptor (LNGFR) or p75 neurotrophin receptor (p75NTR). It can be used in flow cytometry applications for the analysis of cell populations.

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Lab products found in correlation

Cd26 pe, by Miltenyi Biotec (2 mentions) Cd24 pe, by Miltenyi Biotec (2 mentions) Cd34 apc, by Miltenyi Biotec (2 mentions) Cd146 pe, by BD (1 mentions) Igg1 fitc, by BD (1 mentions) Cd90 pe, by Bio-Rad (1 mentions) Cd105 pe, by Bio-Rad (1 mentions) Cd31 fitc, by Bio-Rad (1 mentions) Cellquest software version 3, by BD (1 mentions) Igg1 pe, by Bio-Rad (1 mentions) Facscan, by BD (1 mentions) Cd73 pe, by BD (1 mentions) Nod cg prkdcscidil2rgtm1wjl szj nod scid gamma nsg mice, by Charles River Laboratories (1 mentions) Cd33 apc vio770, by Miltenyi Biotec (1 mentions) Cd3 percp vio700, by Miltenyi Biotec (1 mentions) Cd45 apc, by Miltenyi Biotec (1 mentions) D luciferin, by Ozyme (1 mentions) Pharm lyse, by BD (1 mentions) Caliper ivis lumina 2 system, by PerkinElmer (1 mentions) Facscanto 2, by BD (1 mentions)

8 protocols using cd271 pe

1

Phenotypic profiling of MSCs and ECs

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Phenotypic characterisation was performed on culture expanded MSCs at different passages and on cultured ECs at p4 using: CD31-FITC (#MCA1738F), CD105-PE (#MCA1557PE), CD90-PE (#MCA90PE) (all from Serotec, Kidlington, UK), CD73-PE (#550257), CD146-PE (#550315) (both from BD Pharmingen, Oxford, UK), and CD271-PE (#130-091-885, Miltenyi Biotec). The isotype controls were IgG1-FITC (#550616, BD Pharmingen) and IgG1-PE (#MCA928PE, Serotec). A total of 2×10
5 cells was stained with 5 μl FITC- or PE-conjugated antibodies, and dead cells were excluded using 2 μg/ml propidium iodide (PI, #P1304MP, Invitrogen). Cells were acquired using FACScan equipped with CellQuest software version 3.1 (BD Biosciences) and the proportions of the different fractions were calculated as a percentage of total live cells.
Kouroupis D., Churchman S.M., McGonagle D, & Jones E.A. (2014). The assessment of CD146-based cell sorting and telomere length analysis for establishing the identity of mesenchymal stem cells in human umbilical cord. F1000Research, 3, 126.
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2

Xenograft Mouse Model for CAR T Cell Therapy

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Animal studies were approved by the state animal research committee (LANUV, NRW, Germany) and all animals were cared for according to the guidelines set by the Federation of European Laboratory Animal Science Associations. Six- to 8-week-old female NOD.Cg-PrkdcSCIDIl2rgtm1Wjl/SzJ (NOD-SCID gamma; NSG) mice (Charles River Laboratories, Sulzfeld, Germany) were intravenously engrafted with 3.5 × 106 MOLM-14 cells stably expressing a firefly luciferase-EGFP fusion protein (LucEG). Six days later, mice were intravenously injected with 3.5 × 106 N3-, N4-, or CD8-hinged CD19 or CD33 CAR T cells. At days 6, 13, 20, 27, and 34, the persistence of MOLM-14 cells was assessed via luminescence imaging and PB analysis. For luminescence imaging, mice were intraperitoneally injected with D-luciferin (OZ Biosciences SAS, Marseilles, France) and after 5 min luminescence was measured in a Caliper IVIS Lumina II system (PerkinElmer LAS, Rodgau, Germany) with an exposure time of 15 s. PB was drawn from the tail vein, the erythrocytes lysed with BD Pharm Lyse (BD Biosciences), and the samples analyzed on a MACSQuant Analyzer X flow cytometer for EGFP, CD33, and CD45 expression for MOLM-14 cells and BFP, CAR (ΔNGFR), CD3, and CD45 expression for CAR T cells after staining with CD271-PE, CD3-PerCP-Vio700, CD45-APC, and CD33-APC-Vio770 (all from Miltenyi Biotec)
Bister A., Ibach T., Haist C., Gerhorst G., Smorra D., Soldierer M., Roellecke K., Wagenmann M., Scheckenbach K., Gattermann N., Wiek C, & Hanenberg H. (2022). Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo. Molecular Therapy Oncolytics, 26, 120-134.
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3

Evaluation of CSCs-like Cell Markers

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For the evaluation of CSCs-like cell surface markers, the following fluorochrome-conjugated antibodies were used: CD24-PE, CD26-PE, CD44-PE/CD44-APC, CD133-PE/CD133-APC, CD271-PE, EpCAM-PE (Miltenyi Biotec, Bergisch Gladbach, Germany), CD166-PE (ALCAM, Immunotech, France), and cMET-APC (R&D Systems, Abingdon, UK). Dead cells were excluded from the analysis based on DAPI staining. Cells were analyzed by BD FACSCanto™ II flow cytometer (Beckton Dickinson, USA) equipped with FacsDiva program. FCS Express software was used for the evaluation.
Tyciakova S., Valova V., Svitkova B, & Matuskova M. (2021). Overexpression of TNFα induces senescence, autophagy and mitochondrial dysfunctions in melanoma cells. BMC Cancer, 21, 507.
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4

Multiparameter Flow Cytometry of ASCs

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ASCs, with and without 50% OA-SF, were analyzed by flow cytometry with a CytoFLEX flow cytometer (Beckman Coulter, Fullerton, CA, USA), collecting at least 50,000 events. Antibodies: anti-CD90-FITC (REA897, Miltenyi Biotec, Bergisch Gladbach, Germany), CD73-PE (REA804, Miltenyi), CD105-PerCP-Cy5.5 (43A3, BioLegend, San Diego, CA, USA), CD44-PE-Vio770 (REA690, Miltenyi), CD34-FITC (AC136, Miltenyi), CD271-PE (REA844, Miltenyi), CD31-PerCP-Vio700 (REA730, Miltenyi) and CD45-PE-Vio770 (REA747, Miltenyi). Aggregates were removed from gating events on a FSC-H and FSC-A plot.
Ragni E., Colombini A., Viganò M., Libonati F., Perucca Orfei C., Zagra L, & de Girolamo L. (2021). Cartilage Protective and Immunomodulatory Features of Osteoarthritis Synovial Fluid-Treated Adipose-Derived Mesenchymal Stem Cells Secreted Factors and Extracellular Vesicles-Embedded miRNAs. Cells, 10(5), 1072.
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5

Multiparameter Flow Cytometry Immunophenotyping

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Cells were washed with PBS and stained at room temperature using a 1:10 dilution of the indicated antibodies in 100 µL PBS for 10 min. Cells were washed once more to remove antibody excess and flow cytometry data was subsequently acquired with FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). Live cells were gated based on forward and side scatter plots. Isotype staining’s were performed as control conditions. Anti-CTLA-4 PE extracellular antibody, anti-Gal-9-PE and their corresponding isotype controls were purchased from Biolegend, San Diego, CA, USA. The rest of the antibodies (anti-CD276-PE, anti-CD80-PE, anti-CD86-APC, anti-CD273-PE, anti-CD274-APC, anti-HLA-E-APC, anti-CD112-PE, anti-CD66abce-APC, anti-CD155-PE, anti-CD19-APC or FITC, anti-PD1-PE, anti-TIM3-APC, anti-NKG2A-FITC, CD56-APC, CD96-APC, TIGIT-PE, CD34-APC, CD271-PE and corresponding isotype controls) were obtained from Miltenyi Biotec, Bergisch Gladbach, Germany. CD19-CAR and CD276-CAR expression on NK-92 cells was determined by CD271 and CD34 marker gene expression, respectively, whereas luciferase expression on target cells was correlated with GFP for Nalm-6 GFP/Luc and CD19 marker expression for U-937 CD19tag/Luc.
Ureña-Bailén G., Dobrowolski J.M., Hou Y., Dirlam A., Roig-Merino A., Schleicher S., Atar D., Seitz C., Feucht J., Antony J.S., Mohammadian Gol T., Handgretinger R, & Mezger M. (2022). Preclinical Evaluation of CRISPR-Edited CAR-NK-92 Cells for Off-the-Shelf Treatment of AML and B-ALL. International Journal of Molecular Sciences, 23(21), 12828.
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6

Flow Cytometry Analysis of oBMSC

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For flow cytometry analysis, oBMSC were stained with the following anti-human antibodies: CD34-APC, CD90-FITC, CD73-APC, CD105-PE, CD146-APC, CD271-PE, HLA-DR-PE, and isotype controls (Miltenyi Biotec). Additionally, the following anti-sheep antibodies were used: CD45-FITC and CD44-FITC (Bio-Rad). Cells were stained as per the manufacturers’ instructions, and analysis was completed using an LSR II flow cytometer (BD Biosciences). In this analysis, we also included sheep bone marrow mononuclear cells (MNC) and hBMSC. These cell populations served as controls for antibody performance against a common hBMSC population, and for sheep MNC, where haematopoietic cells (CD45+) would be expected. Data were analysed using FlowJo software, version 10 (BD Biosciences).
Futrega K., Music E., Robey P.G., Gronthos S., Crawford R., Saifzadeh S., Klein T.J, & Doran M.R. (2021). Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo. Stem Cell Research & Therapy, 12, 26.
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7

Immunofluorescence Microscopy Protocol

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IF was performed as reported previously.23 (link) Incubation with primary antibodies CD271-PE (Miltenyi, Bergisch Gladbach, Germany, clone ME20.4-1.H4, mouse IgG1, 1:100), γH2AX (1:250, Cell Signaling Technology), NFκB/p65 and p53 (Cell Signaling Technology, 1:100) or TRITC-labeled phalloidin (1:1000, Sigma-Aldrich) diluted in blocking buffer was done overnight at 4 °C. Second day, cells were washed 3x with phosphate-buffered saline, and incubated with secondary antibodies AlexaFluor488/555 or 594 (1:500) recognizing either rabbit or mouse-produced antibodies and 4′,6-diamidin-2-phenylindol (Sigma-Aldrich, 1:500) for 1 h at room temperature. Washed cells were covered with 500 μl phosphate-buffered saline and used for microscopy. IF pictures were recorded with Zeiss Axiovert40CFL with accompanied Illuminator HPX120C and software AxioVision Rel. 4.8 (all Carl Zeiss AG, Oberkochen, Germany).
Redmer T., Walz I., Klinger B., Khouja S., Welte Y., Schäfer R, & Regenbrecht C. (2017). The role of the cancer stem cell marker CD271 in DNA damage response and drug resistance of melanoma cells. Oncogenesis, 6(1), e291-.
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8

Comprehensive Immunophenotyping of Cell Populations

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Following fluorochrome-conjugated antibodies were used for the evaluation of surface markers: CD44-PE, CD24-PE, CD26-PE, CD271-PE, CD133/2-PE (Miltenyi Biotec, Germany); CD166-PE (ALCAM; Immunotech, France); CD274 (PD-L1; Sony Biotechnology, USA); CD44v6-PE (RD Systems, USA); CD184 (CXCR4-PE; eBioscience, USA). Dead cells were excluded from the analysis based on DAPI staining. Cells were analyzed using BD FACSCanto™ II flow cytometer (Beckton Dickinson, USA) equipped with FacsDiva program. FCS Express software was used for the evaluation.
Durinikova E., Kozovska Z., Poturnajova M., Plava J., Cierna Z., Babelova A., Bohovic R., Schmidtova S., Tomas M., Kucerova L, & Matuskova M. (2018). ALDH1A3 upregulation and spontaneous metastasis formation is associated with acquired chemoresistance in colorectal cancer cells. BMC Cancer, 18, 848.
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