Rabbit polyclone anti-CHOP, cytochrome c (Cyto C), ATF6, PERK, GRP78, ARF4, Bax, Bak, caspase3, cleaved caspase 3, caspase 8, caspase 9, IRE1, DR4, DR5, P-JNK, Bcl-2, Fas, β-Actin, Gapdh, and Tubullin were obtained from Affinity Biosciences (Shanghai, China). Mouse monoclonal anti-Lewis a (Lea), Lewis b (Leb), and Lewis y (Ley) were purchased from Abcam (Cambridgeshire, UK) [74 (link)]. Mouse monoclonal anti-SLex was purchased from BD Biosciences (San Diego, CA, USA) [75 (link)]. FITC-conjugated anti-mouse/rabbit IgG was purchased from Affinity Biosciences (Shanghai, China). Goat anti-rabbit IgG-HRP was obtained from Life Science. E-selectin and P-selectin were purchased from R&D System (Minneapolis, MN, USA). Lectins were purchased from Vector Laboratories (Burlingame, CA, USA). Cells were cultured with cell growth medium (Gibco, Shanghai, China) containing fetal bovine serum (FBS) (ABW, Shanghai, China) and penicillin/streptomycin (Gibco, Shanghai, China).
Lectins
Manufactured by Vector Laboratories
Sourced in United States, Germany, United Kingdom, Sweden
Lectins are a class of carbohydrate-binding proteins found in various organisms. They possess the ability to recognize and bind to specific sugar moieties, allowing them to interact with glycosylated molecules. Lectins are commonly used in scientific research for applications such as cell surface analysis, glycoprotein purification, and histochemical staining.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
P erk, by Affinity Biosciences (1 mentions)
Caspase8, by Affinity Biosciences (1 mentions)
P jnk, by Affinity Biosciences (1 mentions)
Caspase 9, by Affinity Biosciences (1 mentions)
Grp78, by Affinity Biosciences (1 mentions)
Cleaved caspase 3, by Affinity Biosciences (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Caspase 3, by Affinity Biosciences (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hochest 33342, by Thermo Fisher Scientific (1 mentions)
Lncap cell line, by American Type Culture Collection (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Dbco cy3, by Merck Group (1 mentions)
7 protocols using lectins
1
Antibody Procurement for Cell Signaling
2
Cell Line Preparation and Reagent Sourcing
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PNT2 and LNCaP cell lines were obtained from the American Type Culture Collection (ATCC). Lectins were purchased from Vector Laboratories. FeBABE was purchased from Dojindo Molecular Technologies. Dithiothreitol (DTT), iodoacetamide (IAA), DBCO-NH2, and DBCO-Cy3 were purchased from Sigma-Aldrich. Phosphate Buffered Saline (PBS), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, NHS-PEG4-Azide, and Hochest 33342 were purchased from ThermoFisher Scientific. Sequencing Grade Modified Trypsin was purchased from Promega.
3
Flow Cytometry and Immunohistochemistry Analysis
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To analyze the expression of selected surface markers, three-color flow cytometry was used. The cells were stained on ice for 30 min with fluorochrome-conjugated antibodies, then measured on a FACS Calibur flow cytometer (BD Biosciences Immunocytometry Systems, Franklin Lakes, NJ). The data were analyzed using Flowing Software (Cell Imaging Core, Turku Centre for Biotechnology, Finland) and the results were expressed as means of positive cells (%) ± SD. For immunohistochemistry studies, cell cultures were fixed in 4% PFA. Cytoskeletal actin filaments were labelled by phalloidin-TRITC (Sigma-Aldrich, Budapest, Hungary) and the nuclei by Hoechst 33342 (Invitrogen, Oregon, USA). Samples were examined under an Olympus IX81 inverted microscope with MT20 station (Olympus, Münster, Germany) and Orca2 (Hamamatsu Photonics K.K., Japan) camera. Surface carbohydrate molecules were labelled with lectins (Vector Labs, Burlingame, CA) diluted in Hepes buffer (Sigma-Aldrich)and examined by Olympus FluoView 1000 confocal LSM (Olympus).
4
Phosphatidylserine and Glycan Exposure
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Human cervical adenocarcinoma HeLa cells and human colorectal epithelial Caco-2 and T84 cells were obtained from the American Type Culture Collection (ATCC® CCL-2, HTB-37, CCL-248, respectively) and cultured in RPMI-1640 and Eagle's Minimum Essential Medium (EMEM), respectively (Sigma Chemical Co., USA). The media were supplemented with 4 mM L-glutamine, 10 mM HEPES buffer, 100 U/mL penicillin, 100 μg/mL streptomycin (PAA, Pasching, Germany), and fetal calf serum (Gibco-BRL, Eggenstein, Germany) at 20% (v/v) for Caco-2 cells and 10% for HeLa cells. Cultures were maintained under 5% CO2 at 37°C.
To detect surface exposure of phosphatidylserine and glycan, cells were stained with FITC-conjugated Annexin V (AxV-FITC, Böhringer, Mannheim, Germany) and lectins (Vector Laboratories). The cells were grown on slides of 22 × 22 mm, washed twice with Ringer's solution, and placed in 200 μL of reaction medium (Ringer's solution with 100 ng of AxV-FITC Nicoletti et al., 1991 (link) or 1–5 μg/mL of lectins). The samples were analyzed immediately by fluorescence microscopy.
To detect surface exposure of phosphatidylserine and glycan, cells were stained with FITC-conjugated Annexin V (AxV-FITC, Böhringer, Mannheim, Germany) and lectins (Vector Laboratories). The cells were grown on slides of 22 × 22 mm, washed twice with Ringer's solution, and placed in 200 μL of reaction medium (Ringer's solution with 100 ng of AxV-FITC Nicoletti et al., 1991 (link) or 1–5 μg/mL of lectins). The samples were analyzed immediately by fluorescence microscopy.
5
Colon Tissue Histological Analysis
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Mice were euthanized with CO2 and death was confirmed by cervical dislocation. After sacrifice, sections of the intestinal tract (colon) were fixed using paraformaldehyde. They were then embedded, sectioned, and stained as previously described 70 . Briefly, two sections per mouse were fixed in 2% paraformaldehyde in phosphate buffer for 16-18 h, washed in 70% ethanol, and then paraffin-embedded. Samples were sectioned into 4 µmthick sections for staining with DAPI, fluorescence in situ hybridization probes targeting Gammaproteobacteria (Gam42A: 5'-GCCTTCCCACATCGTTT-3') and E. coli (Eco1167: 5'-GCATAAGCGTCGCTGCCG-3') 71 , and lectins targeting mucin carbohydrates (Vector Laboratories, Burlingame, CA). Fluorescence in situ hybridization was performed as previously described 70 . Sections of the distal colon were imaged on a Zeiss LSM 880 confocal microscope equipped with an Airyscan super-resolution module. Airyscan images were processed using Zen Blue and FIJI.
6
Lectin Microarray Analysis of CD4+ T Cells
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The lectin microarray was prepared as previously described [35, 36] . Briefly, lectins (Vector Laboratories, Inc., Burlingame, CA) (Table 1) were dissolved in spotting solution at a concentration of 1 mg/ mL (Sambucus nigra leptin, MAL-I, and MAL-II) or 0.5 mg/mL (other lectins) and spotted in duplicate onto a 3-glycidoxypropyltrimethoxysilane glass slide using a microarray printing robot (MicroGridII; BioRobotics Ltd., Cambridge, UK). After being spotted, washed, and incubated, the slides were blocked with 1% bovine serum albumin. Purified CD4 + T cells from the spleens of WT and Glt25d2 -/-mice were cultured in RPMI 1640 media. Before and after Con A treatment for 6, 12, or 24 h, the harvested CD4 + T cells were suspended in PBS and allowed to bind to lectin microarrays at 37°C for 30 min. The unbound CD4 + T cells were washed with cold PBS. The bound CD4 + T cells immobilized on the glass slides were stained with H&E and observed by microscopy.
7
Immunoassay Reagents and Protocols
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Reagents used were Carlo Erba Reagents S.A.S or Merck/Sigma chemicals co. Molecular standards and nitrocellulose strips were purchased to GE Healthcare, Uppsala, Sweden.
Lectins, streptavidin conjugate and ABC system for electrochemiluminescence were Vector Labs, Burlingame, CA, USA. Goat anti-rabbit horseradish-peroxidase conjugated secondary antibody used was bought to BioRad Laboratories, Inc. Alexa Fluor 488 Protein Labelling kit was Invitrogen, Life Technologies-Molecular Probes. U-shaped microtiter plates for hemagglutinating assays were Greiner Bio One, Germany.
Lectins, streptavidin conjugate and ABC system for electrochemiluminescence were Vector Labs, Burlingame, CA, USA. Goat anti-rabbit horseradish-peroxidase conjugated secondary antibody used was bought to BioRad Laboratories, Inc. Alexa Fluor 488 Protein Labelling kit was Invitrogen, Life Technologies-Molecular Probes. U-shaped microtiter plates for hemagglutinating assays were Greiner Bio One, Germany.
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