A647 anti-MICA/B, APC anti-CD3, FITC anti-CD56, APC anti-HLA-E, PE anti-HLA-A/B/C, blocking anti-NKG2D and all isotype control were from Biolegend. PE anti-MICA, -MICB, ULBP1, -ULBP2 and -ULBP3 were from R&D systems. FITC anti-pan γδTCR was from Beckman Coulter. PE anti-CD107a was from BD. Cetuximab (Erbitux) was from Merck. Recombinant human EGF was from Calbiochem. All cell culture media and reagents, Alexa 488 goat anti-rabbit IgG, and Prolongold were from Invitrogen. Chemical inhibitors and rabbit polyclonal anti-AUF1 and anti-phosphoAUF1 were from Sigma-Aldrich. The Dual Luciferase reporter system was from Promega.
Ulbp3
Manufactured by R&D Systems
Sourced in Germany
ULBP3 is a lab equipment product offered by R&D Systems. It is a compact and versatile device designed for performing various laboratory functions. The core function of ULBP3 is to provide a reliable and consistent platform for conducting scientific experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ulbp1, by R&D Systems (4 mentions)
Ulbp2, by R&D Systems (3 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Cellquest software, by BD (2 mentions)
Ulbp1 pe, by R&D Systems (2 mentions)
Hla abc pe, by BD (2 mentions)
Hla g fitc, by Bio-Rad (2 mentions)
Mica micb fitc, by Bio-Rad (2 mentions)
Cd155 fitc, by Bio-Rad (2 mentions)
Ulbp 2 pe, by R&D Systems (2 mentions)
Cd58 pe, by BD (2 mentions)
Tlr9 fitc, by Abcam (2 mentions)
Tlr4 pe, by Abcam (2 mentions)
Hla e, by Abcam (2 mentions)
Cd112, by Bio-Rad (2 mentions)
Ulbp2 5 6, by R&D Systems (2 mentions)
Alexa 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti cd107a pe, by BD (1 mentions)
Anti cd56 fitc, by BioLegend (1 mentions)
Apc anti hla e, by BioLegend (1 mentions)
7 protocols using ulbp3
1
Immune Response Modulation Assay
2
FACS Analysis of NKG2D Ligands
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Fluorescence-activated cell sorting (FACS) was performed on a FACSCalibur (Becton Dickinson, Heidelberg, Germany) or Gallios (Beckman Coulter, Krefeld, Germany). 7-Aminoactinomycin D (7-AAD; BioLegend, Koblenz, Germany) was added before measurement and only negative cells were included in analysis. The following specific antibodies were used: ac-Lysine (9441, CST, Leiden, Netherlands); MICA (AMO1, BamOmaB); MICA/B (6D4, BioLegend); MICB (BMO2, BamOmaB, Gräfelfing, Germany); MULT-1 (237104, R&D); p-γH2AX (S139, 9718, CST); Rae-1 (R&D, Wiesbaden-Nordenstadt, Germany); ULBP1 (AUMO2, BamOmaB); ULBP2 (BUMO1, BamOmaB); ULBP2 (BAF1298, R&D); ULBP3 (CUMO3 BamOmaB) and isotype controls purchased from BioLegend.
3
Evaluating Surface Marker Expression in MSC Variants
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The expression of surface markers on MSC, nvMSC-HLA-G1, and vMSC-HLA-G1 was evaluated by flow cytometry using the following monoclonal anti-human antibodies: HLA-G FITC (ABD Serotec, Oxford, UK), HLA DR, DP, DQ FITC (BD Bioscience, San Jose, CA), HLA-ABC PE (BD Pharmingen, San Jose, CA), MICA/MICB FITC, CD155 FITC (both from ABD Serotec), ULBP-1 PE, ULBP-2 PE (both from R&D Systems), CD58 PE (BD Biosciences), TLR9 FITC, TLR4 PE (both from Abcam, Cambridge, UK) and TLR3 Alexa 647 (Imagenex, Port Coquitlam, BC, Canada). Cells were also stained for HLA E (Abcam), ULBP-3 (R&D Systems), and CD112 (ABD Serotec) using Alexa Fluor 488 Goat Anti-Mouse IgG (Molecular Probes-Life Technologies) as secondary antibody. Briefly, cells were incubated for 15 minutes in the dark with saturating concentrations of each antibody. Stained cells were then washed with phosphate-buffered saline 0.1% azide, fixed with 1% formaldehyde, and analyzed by flow cytometry using the CellQuest software (Becton Dickinson Biosciences, San Jose, CA). A total of 10,000 events was acquired for each sample. Appropriate isotype (negative) controls were performed for each antibody.
4
Phenotyping Cervical Cancer Cell Lines
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To phenotype cervical cancer cell lines, cell suspensions in PBS supplemented with 0.1 % BSA and 0.02 % NaN3 (FACS buffer) were stained for 30 min at 4 °C using antibodies to HLA-ABC (clone w6/32, Immunotools) (labeled with FITC), HLA-E (clone 3D12HLA-E, eBioscience), HLA-G (clone 87G, Biolegend), EGFR (clone EGFR.1, BD Biosciences), PVR (clone SK11.4, Biolegend), MICA/B (clone 6D4, Biolegend), ULBP2/5/6 (clone #165903, R&D systems), ULBP1 (clone #170818, R&D systems) and ULBP3 (clone #166510, R&D systems) (all labeled with PE). IgG1, IgG2a and IgG2b isotype antibodies were used as negative controls. After incubation, the cells were washed with FACS buffer and analyzed using a flow cytometer LSR Fortessa (BD Biosciences). Phenotypic analyses were obtained from at least two independent experiments performed on each cell line. Data were analyzed using Kaluza software (Beckman coulter) and calculated as specific (geometric) mean fluorescence intensity (MFI) (MFI; geometric mean fluorescence of marker − geometric mean fluorescence of isotype).
5
Quantitative MICA, MICB, ULBP Assay Protocol
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ELISAs for MICA, MICB, ULBP1, ULBP2, and ULBP3 (R&D) were performed as per the manufacturer's instructions. All samples and standards were assayed in duplicate. Plates were read immediately using an automated plate reader (Finstruments Multiskan) at 450 nm. Mean optical densities (OD) for each standard and sample were taken and antigen concentrations were calculated using Excel for Mac 2011 (Microsoft).
6
Immunophenotyping of γδ T cells and Breast Cancer Cells
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For surface marker staining of γδ T cells, the following anti-human antibodies from BioLegend (unless otherwise indicated) were employed: TCRγδ PE (clone B1, 1:25); TCRγδ PE (Miltenyi, clone REA591, 1:10); TCRγδ BV421(clone B1, 1:10); TCR Vδ1 FITC (Miltenyi, clone REA173, 1:10); TCR Vδ2 PE (Miltenyi, clone 123R3, 1:100); TCR Vδ2 PerCP (clone B6, 1:25); CD27 AF700 (clone M-T271, 1:25); CD27 APC (clone M-T271, 1:25); CD45RA FITC (clone HI100, 1:25); CD69 AF700 (clone FN50, 1:4); CTLA-4 APC (clone L3D10, 5 μl); and PD-1 BV421 (clone EH12.2H7, 1:20).
For breast cancer cell line surface staining, anti-human MICA/B PE (clone 6D4, 0.1 μg); ULBP-2,5,6 (R&D systems, clone 165,903, 0.2 μg); ULBP-3 (R&D systems, clone 166,510, 0.04 μg); ULBP-4 (R&D systems, clone 709,116, 0.1 μg).
For breast cancer cell line surface staining, anti-human MICA/B PE (clone 6D4, 0.1 μg); ULBP-2,5,6 (R&D systems, clone 165,903, 0.2 μg); ULBP-3 (R&D systems, clone 166,510, 0.04 μg); ULBP-4 (R&D systems, clone 709,116, 0.1 μg).
7
Evaluating Surface Marker Expression in MSC Variants
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