The differentiation state of HLEC was determined using immunofluorescence to assess expression of the putative LESC markers ABCG2 and p63alpha and the corneal epithelium differentiation marker CK3 (cytokeratin 3). HLEC were cultured in 4-well chamber slides and labeled with Qdots. Slides containing unlabeled cells were used as negative controls. Five days after labeling, cells were washed, fixed with 3.2% paraformaldehyde (ABCG2/CK3) or methanol (p63alpha) for 5 minutes at room temperature, washed with PBS containing 0.5% triton X-100 (Sigma), and blocking of nonspecific primary antibody binding was performed using 10% goat serum in PBS for 30 minutes. Incubation with primary antibodies to ABCG2 (Chemicon), p63alpha (Cell Signalling Technologies) and CK3 (Santa Cruz Biotechnologies) was carried out overnight at 4°C. The slides were washed and incubation with the secondary antibody was performed at room temperature for 2 hours. Slides were then washed and mounted using Vectashield containing DAPI (Vector Labs). Slides were examined and images collected using a Zeiss LSM 510 confocal microscope.
Abcg2
Manufactured by Merck Group
Sourced in United States
ABCG2 is a laboratory equipment product from Merck Group. It is a cell membrane transporter protein that functions as an ATP-binding cassette (ABC) efflux pump. ABCG2 is involved in the regulation of various cellular processes.
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Lab products found in correlation
Lsm 510 confocal microscope, by Zeiss (2 mentions)
Vectashield containing dapi, by Vector Laboratories (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Snail, by Santa Cruz Biotechnology (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Ubiquitin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vascular endothelial growth factor (vegf), by ABclonal (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Cxcr4, by R&D Systems (1 mentions)
Cd117, by BD (1 mentions)
Cd133, by Miltenyi Biotec (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Hla dr, by BD (1 mentions)
Cd271, by BD (1 mentions)
Integrin β4, by Abcam (1 mentions)
Elisa assay, by Cusabio (1 mentions)
Zonula occludens 1, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
6 protocols using abcg2
1
Characterizing HLEC Differentiation State
2
Immunoblotting and Immunoprecipitation Assays for Hypoxia Signaling
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IP lysis buffer with a protease inhibitor cocktail was used to harvest the protein from the cells. 40 μg of total protein was transferred onto a PVDF membrane. The membrane was subjected to immunoblotting by incubating with specific primary antibodies overnight at 4°C. Antibody signals were detected by ChemiDox XRS+.
For immunoprecipitation experiments, cells were harvested, lysed, quantified, and precleared with 20 μL of Dynabeads™ Protein G (ThermoFisher Scientific, #10004D, USA). After centrifugation, the supernatant was collected and incubated with antibody at 4°C overnight. Dynabeads™ Protein G was added to the protein‐antibody complex for additional incubation. Subsequently, the beads were collected and boiled. Western blot analysis was performed on the samples, with 20 μg of total protein used as the input control.
The antibodies for Western Blot: HIF‐1α (CST, #36169S, 1:1000), Hydroxylation HIF‐1α (CST, #3434T, 1:1000), PHD3 (Novus, #NB100‐139, 1:1000), GLUT1 (CST, #12939S, 1:1000), RP58 (Proteintech, #12714‐1‐AP, 1:1000), PHD2 (CST, #4835S, 1:1000), VEGF (Abclonal, #A12303, 1:500), N‐cadherin (CST, #13116, 1:1000), HK2 (Proteintech, #22029‐1‐AP, 1:2000), Ubiquitin (CST, #3936S, 1:1000), PGK1 (Proteintech, #17811‐1‐AP, 1:1000), β‐actin (Sigma, #A5441, 1:10000), ABCG2 (Sigma‐Aldrich, MAB4146, 1:1000), and anti‐Flag (CST, #14793, 1:1000).
For immunoprecipitation experiments, cells were harvested, lysed, quantified, and precleared with 20 μL of Dynabeads™ Protein G (ThermoFisher Scientific, #10004D, USA). After centrifugation, the supernatant was collected and incubated with antibody at 4°C overnight. Dynabeads™ Protein G was added to the protein‐antibody complex for additional incubation. Subsequently, the beads were collected and boiled. Western blot analysis was performed on the samples, with 20 μg of total protein used as the input control.
The antibodies for Western Blot: HIF‐1α (CST, #36169S, 1:1000), Hydroxylation HIF‐1α (CST, #3434T, 1:1000), PHD3 (Novus, #NB100‐139, 1:1000), GLUT1 (CST, #12939S, 1:1000), RP58 (Proteintech, #12714‐1‐AP, 1:1000), PHD2 (CST, #4835S, 1:1000), VEGF (Abclonal, #A12303, 1:500), N‐cadherin (CST, #13116, 1:1000), HK2 (Proteintech, #22029‐1‐AP, 1:2000), Ubiquitin (CST, #3936S, 1:1000), PGK1 (Proteintech, #17811‐1‐AP, 1:1000), β‐actin (Sigma, #A5441, 1:10000), ABCG2 (Sigma‐Aldrich, MAB4146, 1:1000), and anti‐Flag (CST, #14793, 1:1000).
3
Comprehensive Immunophenotypic Characterization of Cultured Cells
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At passage 3 (P3), cells grown in expansion medium were detached with 0.25% trypsin-EDTA (Sigma-Aldrich) and, after a 20 minutes recovery phase, were incubated with the following properly conjugated primary antibodies: CD10, CD13, CD29, CD49a, CD49b, CD49d, CD90, CD73, CD44, CD59, CD45, HLA-DR, CD117, CD34, CD 271 (BD Biosciences), CD105, CD66e, KDR (Serotech), CD133 (Miltenyi Biotec), CXCR4 (R&D), ABCG-2 (Chemicon International).
4
Corneal Epithelial Differentiation Analysis
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The differentiation phenotype was assessed with immunofluorescence staining (n = 4) for K3 (1∶100, Millipore), P63 (1∶50, Millipore) and ABCG2 (1∶50, Millipore). Proliferative activity was measured via colony-forming efficiency (n = 10), as previously described [31] (link). Briefly, the epithelium of each specimen was trypsinized and planted at a density of 500 cells per 60 mm in dishes that had been preseeded with 3T3 fibroblast feeder layers. Colony-forming efficiency was calculated on day 6. Cell junction-related proteins zonula occludens-1 (1∶50, Invitrogen), desmocollin 2 (1∶100, Abcam), and integrin β4 (1∶50, Abcam) were detected using immunofluorescence staining (n = 4). Then, the contents of these proteins were quantitated using ELISA assays according to the manufacturer’s recommendations (n = 10, Cusabio Biotech). The ultrastructures of cell junctions in each group were observed using TEM (n = 4).
5
Western Blot Analysis of Stem Cell and Cancer Markers
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Whole cell lysates (50 μg) were separated by electrophoresis on 12.5% denaturing polyacrylamide gels. The membranes were incubated overnight at 4°C with primary antibodies (0.1 μg/ml) against Oct4, Nanog, β-AR and phospho EGFR were purchased from Abcam Corporation (Abcam, Cambridge, UK), Snail, Twist, Fibronectin, E-cadherin, CD21, CD44, CD133, ALDH-1, phospho AKT, phospho NFκB, α7-nAChR, Bcl-2 were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Bax was obtained from Becton, Dickinson and Company (BD, Transduction Laboratories TM), MDR-1 and ABCG2 were purchased from Millipore Corporation (Millipore Corporation, Billerica, MA, USA), caspase-3 and PARP were obtained from Invitrogen Corporation (Invitrogen, Camarillo, CA, USA) in Tris-Tween-Buffer-Saline buffer containing 3% non-fat milk. Subsequently, each membrane was washed and incubated for 1 h at 25°C with a secondary anti-mouse, anti-rabbit, or anti-goat antibody conjugated with horseradish peroxidase (1: 1000; Santa Cruz Biotechnology, Inc.), as previously reported. [25 (link)]
6
Western Blot Analysis of Stemness Markers
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Western blot was performed following the standard protocol [22 (link), 31 (link)]. The cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to the polyvinylidene fluoride membranes. After the membranes were blocked, the diluted primary antibodies, MDR1 (MAB4163, 1:1000, Chemicon, Temecula, CA, USA ), ABCG2 (MAB4155, 1:1000, Millipore, Billerica, MA, USA), α-Tubulin (sc-5286, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (sc-7870, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Snail (sc-28199, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nanog (sc-81961, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Oct4 (GTX101497, 1:1000, Gene Tex, San Antonio, TX, USA), in Tris-buffered saline with Tween (TBST) buffer containing 3% non-fat milk were added to the membranes and incubated at 4°C overnight. On the second day, the goat anti-mouse conjugated secondary antibody was added at room temperature for 1 h. The immunoblots were developed using an enhanced chemiluminescence system and visualized on X-ray film.
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