Cells were cultured in a glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) and fixed with 4% paraformaldehyde for 10 min at 4 °C before being permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature (RT). The samples were blocked with 5% normal goat serum in Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific) for 30 min at RT. They were incubated with primary antibodies at 4 °C overnight. After washing with DPBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 or 546 (Thermo Fisher Scientific) for 30 min at RT. After washing with DPBS, the samples were placed in mounting medium with 4',6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: OCT4 (C-10; Santa Cruz Biotechnology, Dallas, TX, USA), NANOG (ReproCell), Tra1-60 (MAB4360; Sigma-Aldrich), SSEA4 (MAB1435; R&D Systems), SSEA1 (MAB2155; R&D Systems), KLF4 (ab216875; Abcam), PRDM14 (ab187881; Abcam), anti-βIII Tubulin (TUJ1, Promega, Madison, Wisconsin, USA), α-smooth muscle actin (α-SMA; A2547; Sigma-Aldrich), and SOX17 (MAB1924; R&D Systems). Images were acquired using an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). All antibodies except for the anti-TUJ1 antibody (1:300) were used at a 1:150 dilution in 5% normal goat serum.
Mab2155
Manufactured by R&D Systems
8 citations
Sourced in United States
About the product
MAB2155 is a monoclonal antibody that targets the human IL-1 beta protein. It is intended for use in research applications.
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8 protocols using «mab2155»
1
Immunofluorescence Staining of Stem Cells
Corresponding organizations : Teikyo University, National Center For Child Health and Development, University of Southern California, The University of Tokyo
2
Characterizing Pluripotent Stem Cells
For immunofluorescent staining, reprogramming was carried out on coverslips coated with 0.1% gelatin. Antibodies against SSEA1 (MAB2155; R&D systems) and Nanog (AF1997; R&D) were applied at a dilution of 1:500. Alkaline phosphatase staining was performed using an alkaline phosphatase detection kit (Millipore) according to the manufacturer’s instructions.
Corresponding organizations : Shenyang Medical College, University Medical Center Hamburg-Eppendorf, Universität Hamburg, Mayo Clinic
3
Pluripotent Stem Cell Characterization
Established pluripotent (beyond passage 15) DF‐iPSCs and ACL‐iPSCs were seeded into 24‐well plates and grown for 4 days. Pluripotent cells and outgrown EBs were fixed with 4% paraformaldehyde in PBS for 10 min. Cells were then incubated with primary antibodies against markers of pluripotency; NANOG (1:400, cat. no. 4903; Cell Signaling Technology, London, UK), OCT‐4 (1:100, cat. no. 611202; BD Biosciences, Oxford, UK), SOX2 (1:400, cat. no. 3579; Cell Signaling Technology), SSEA‐3 (1:200, cat. no MAB1434; R&D Systems, Abingdon, UK), SSEA‐4 (1:200, cat. no MAB1435; R&D Systems), TRA‐1‐60 (1:200, cat. no. Ab16288; Abcam, Cambridge, UK), TRA‐1‐81 (1:200, cat. no. Ab16289; Abcam), marker of early differentiation; SSEA‐1 (1:200, cat. no. MAB2155; R&D Systems), marker of mesoderm; α‐smooth muscle actin (αSMA) (1:100, cat. no. MAB1420; R&D Systems), marker of endoderm; GATA6 (1:1600, cat. no. 5851; Cell Signaling Technology) and marker of ectoderm; Neurofilament (1:100, cat. no. 2837; Cell Signaling Technology), in the presence of 1% goat serum, followed by Alexa Fluor secondary antibodies (1:200; Thermo Fisher Scientific) and nuclei stained using 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat no. D1306; Thermo Fisher Scientific). Images were captured using BX51 fluorescence microscope (Olympus, Southend‐on‐Sea, UK).
Corresponding organizations : University of Manchester, Wellcome Centre for Cell-Matrix Research, Manchester University NHS Foundation Trust
4
Characterizing Hypoxia-Induced Stemness Markers
Sorted cells were cultured under hypoxia for 48 h to detect the expression of CD133, SOX-2, OCT-4, Lin-28A, KLF-4, Nanog, CD15 and NESTIN. The sorted cell lines were exposed to hypoxia for 48 h, and 4% paraformaldehyde was used to fix the cells at 4 °C for 10 min. The cells were washed with PBS, and 10% serum in PBS containing 0.5% Triton X-100 was used to block the cells. The cells were then incubated for 24 h at 4 °C with primary antibodies against CD133 (1 : 150, MBS462020, MyBiosource, San Diego, CA, USA), SOX-2 (1 : 100, MAB2018, R&D Systems), KLF-4 (1 : 100, Human: AF3640; Mouse: AF3158; R&D Systems), OCT-4 (1 : 100, MAB1759 R&D Systems), Nanog (1 : 100, Human: AF1997; Mouse: AF2729 R&D Systems), Lin-28A (1 : 100, NBP1–49537, Novus Biologicals), CD15 (1 : 100, MAB2155, R&D Systems) or NESTIN (1 : 100, MAB2736, R&D Systems). The cells were then washed with PBS three times. Appropriate fluorophore-labeled secondary antibodies were added to the cells and incubated at 37 °C for 1 h. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Jena, Thuringia, Germany).
Corresponding organizations : Army Medical University, Southwest Hospital, Chongqing Cancer Hospital
5
Hypoxia-Induced Stem Cell Marker Analysis
Proteins were collected from sorted cells after hypoxia exposure for 0, 12, 24, 48 and 72 h. We then subjected the proteins to SDS-PAGE and transferred them to nitrocellulose membranes, which were blocked with 5% non-fat milk and incubated with primary antibodies against CD133 (1 : 1000, MBS462020, MyBiosource, San Diego, CA, USA), SOX-2 (1 : 1000, MAB2018, R&D Systems, Minneapolis, MN, USA), KLF-4 (1 : 1000, Human: AF3640; Mouse: AF3158 R&D Systems), OCT-4 (1 : 1000, MAB1759, R&D Systems), Nanog (1 : 1000, Human: AF1997; Mouse: AF2729 R&D Systems), Lin-28A (1 : 1000, NBP1–49537, Novus Biologicals, Littleton, CO, USA), CD15 (1 : 1000, MAB2155, R&D Systems) or NESTIN (1 : 1000, MAB2736, R&D Systems). β-Actin was used as an internal control.
Corresponding organizations : Army Medical University, Southwest Hospital, Chongqing Cancer Hospital
Top 3 most cited protocols using «mab2155»
1
Immunophenotyping of Hypoxic Neurospheres
Newly formed neurospheres and differentiated cells after 48 h of hypoxia were fixed with 4% paraformaldehyde for 10 min at 4°C, washed with PBS and blocked with 10% normal serum for 20 min in PBS that contained 0.5% Triton X-100. The cells were incubated for 24 h at 4°C with primary antibodies against SOX-2 (1:100, MAB2018, R&D Systems, USA), OCT-4 (1:100, MAB1759, R&D Systems, USA), Nanog (1:100, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:100, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:150, MBS462020, MyBiosource, USA), CD15 (1:100, MAB2155, R&D Systems, USA), NESTIN (1:100, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:100, ab130244, Abcam, USA), VEGF (1:100, MAB293, R&D Systems, USA) and HIF1α (1:100, MAB1536, R&D Systems, USA). Neurospheres and cells were washed three times with PBS for 5 min and then incubated at 37°C for 1 h with appropriate fluorophore-labeled secondary antibodies. Images were acquired with a laser scanning confocal microscope (LSM780, ZEISS, Germany).
Corresponding organizations : Army Medical University, Southwest Hospital, Chongqing Cancer Hospital, Xinqiao Hospital
2
Antibody Production and Purification
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Corresponding organizations : Zhejiang University, Institute of Genetics and Developmental Biology, The Ohio State University, Nationwide Children's Hospital
3
Hypoxia-Induced Stemness Marker Expression
Cells cultured in hypoxia for 0, 12, 24, 48 and 72 h were collected, subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and incubated with antibodies against SOX-2 (1:1000, MAB2018, R&D Systems, USA), OCT-4 (1:1000, MAB1759, R&D Systems, USA), Nanog (1:1000, Human: AF1997; Mouse: AF2729, R&D Systems, USA), KLF-4 (1:1000, Human: AF3640; Mouse: AF3158, R&D Systems, USA), CD133 (1:1000, MBS462020, MyBiosource, USA), CD15 (1:1000, MAB2155, R&D Systems, USA), NESTIN (1:1000, Human: MAB1259; Mouse: MAB2736, R&D Systems, USA), ABCG2 (1:1000, ab130244, Abcam, USA), VEGF (1:1000, MAB293, R&D Systems, USA) and HIF1α (1:1000, MAB1536, R&D Systems, USA). Enhanced chemiluminescence was conducted for visualization.
Corresponding organizations : Army Medical University, Southwest Hospital, Chongqing Cancer Hospital, Xinqiao Hospital
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