Flow cytometry was performed by staining with Zombie Yellow viability dye, blocking with anti-CD16/32, and staining for 30 min at 4°C with the following anti-human antibodies: CD45 (BD:HI30), CD3 (BD:UCHT1), CD11b (Biolegend:M1-70), CD206 (Biolegend:15-2), CD163 (Biolegend:GHI/61), CD271 (Biolegend:ME20.4), PD-L1 (Biolegend:29E.2A3); or anti-mouse antibodies: CD45 (Biolegend:30-F-11), CD11b (Biolegend:M1-70), F4/80 (Biolegend:F4/80), Ly6G (Biolegend:1A8), Ly6C (Biolegend:HK1.4), IA/IE (BD:M5/114), CD206 (Biolegend:C068C2), PD-L1 (Biolegend:10F.9G2), PD-1 (BD:J43), IFN-γ (Biolegend:XMG1.2), CD3 (Tonbo:145-2C11), CD8 (Biolegend:YTS156.7.7), CD4 (BD:GK1.5), CD106 (Biolegend:429-MVCAM.A), EpCAM (Biolegend:G8.8), CD44 (Biolegend:IM7), Sca1 (Biolegend:D7). For IFN-γ, cells were pre-stimulated with PMA (20 ng/mL), Ionomycin (Sigma, 1 μg/mL) and Golgi stop (BD, 0.8 μL/106 cells) for 4 hr. Samples were subsequently run using BD FACS LSRII or sorted using BD FACS ARIA. Data were analyzed using FlowJo.
Cd271
Manufactured by BioLegend
Sourced in United States
CD271, also known as nerve growth factor receptor (NGFR) or p75 neurotrophin receptor, is a transmembrane glycoprotein that functions as a receptor for neurotrophins. It is expressed on various cell types, including neurons, Schwann cells, and some immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Pd l1, by BioLegend (2 mentions)
Cd11b, by BioLegend (2 mentions)
F4 80, by BioLegend (2 mentions)
Aria 3, by BD (2 mentions)
Anti cd133 pe, by BioLegend (2 mentions)
Facs lsr 2, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Sca 1, by BioLegend (1 mentions)
Cd163, by BioLegend (1 mentions)
I a 1 e, by BD (1 mentions)
Cd106, by BioLegend (1 mentions)
Epcam, by BioLegend (1 mentions)
Ifn γ, by BioLegend (1 mentions)
Cd206, by BioLegend (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Interleukin 2 (il 2), by BioLegend (1 mentions)
Tnf α, by BD (1 mentions)
Tnf α, by BioLegend (1 mentions)
8 protocols using cd271
1
Multi-parameter Flow Cytometry Profiling
2
Comprehensive Antibody Panel for Immune Cell Analysis
3
Directed Differentiation of hiPSCs into iMSCs
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The differentiation method for hiPSC-derived iMSCs was applied as described previously.27 (link) Briefly, as pre-induction, hiPSCs were cultured in an mTESR1 medium (STEMCELL Technologies, Vancouver, BC, Canada) in cell culture plates coated with growth factor-reduced Matrigel (Corning) for a few days. Post preincubation, hiPSCs were differentiated into iNCCs by replacing the mTESR1 medium with a chemically defined medium (CDM)95 (link) supplemented with 10 μM SB431542 (Sigma-Aldrich, St. Louis, MO, USA) and 1 μM CHIR99021 (Fujifilm Wako, Osaka, Japan). CDM contained Iscove’s modified Dulbecco’s medium/Ham’s F-12 1:1, 1× chemically defined lipid concentrate (Gibco, Grand Island, NY, USA), 15 μg/mL apo-transferrin (Sigma-Aldrich), 450 μM monothioglycerol (Sigma-Aldrich), 5 mg/mL purified BSA (99% purified by crystallization, Sigma-Aldrich), 7 μg/mL insulin (Fujifilm Wako), and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Differentiated hiPSCs were subsequently sorted for CD271 (BioLegend, San Diego, CA, USA) (iNCCs). iNCCs were maintained in CDM supplemented with SB431542, 20 ng/mL epidermal growth factor (EGF) (R&D Systems, Minneapolis, MN, USA), and 20 ng/mL FGF2 (Fujifilm Wako).96 (link)
4
Isolation of Uncultured Cells from Breast Organoids
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For dissociation of uncultured cells from organoids, organoids were digested with 0.5% trypsin/EDTA for 10 minutes at 37°C with agitation. After trypsin treatment, organoids were disrupted by vigorous shaking for 30 seconds. Then, cells were passed through 40 μm cell strainer (BD Falcon). Dissociated uncultured cells from breast organoids and fourth-passage HMEC were stained with anti-CD133-PE (BioLegend, clone 7) and CD271 (BioLegend, clone ME20.4) by following the standard flow cytometry protocol. Cells were sorted by AriaIII (Becton Dickinson).
5
Dissociation and Sorting of Organoid Cells
6
Phenotypic characterization of MSCs
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MSC of passages 2 were labelled with fluorochrome-conjugated mouse anti-human antibodies against MSCs antigens CD90, CD73, CD105, CD146, CD271 (BioLegend, Koblenz, Germany) and hematopoietic antigens CD45, CD34 and CD14 as well as HLA Class II molecules (HLA-DR) (BioLegend, Koblenz, Germany). MSCs were incubated at 4 °C for 30 min, and after two wash steps with PBS + 0.2% bovine serum albumin BSA, flow cytometric analysis was performed on a FACSCalibur (Becton-Dickinson, Heidelberg) equipped with Macintosh software for data analysis (CellQuest). At least 50,000 events were acquired for each measurement.
7
Melanoma Cell Protein Profiling
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Whole-cell lysates from SK-MEL-28 cells and primary melanoma cells were processed as previously described [23 (link)]. Primary antibodies were raised against the following molecules: actin and Nox4 (Sigma-Aldrich, St. Louis, MO, USA), phospho AktS473, phospho AktT308, phospo ERK, Akt1, Akt2, Akt3, and ERK (Cell Signaling Technology, Lieden, Netherlands), Vimentin, ZEB1, and Slug (Santa Cruz Biotechnology, Dallas, TX, USA), and CD271 (BioLegend, San Diego, CA, USA).
Secondary antibodies, used at 1:3000 dilutions, were all obtained from Thermo Fisher Scientific (Waltham, MA, USA).
Secondary antibodies, used at 1:3000 dilutions, were all obtained from Thermo Fisher Scientific (Waltham, MA, USA).
8
CAR T Cell Activation and Function
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CAR T cells were incubated alone or with UM9 cells, in the presence or absence of BMMSCs for 24 hours. Mosenin (BioLegend) was added 4 hours prior to termination of the assay. Nonadherent cells were harvested by gentle pipetting and stained for LNGFR (CD271, BioLegend), CD3 (BD), and live/dead marker (LIVE/DEAD Fixable Near-IR; Life Technologies). The cells were fixed in 4% formaldehyde for 5 minutes at room temperature (RT). After fixation, the cells were placed in FACS lysing solution (BD) for 10 minutes at RT. The permeabilized cells were stained for IFNg and granzyme B (BioLegend) for 30 minutes at RT. Expression
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