Untouched human basophils were stripped of pre-bound endogenous IgD as described in published studies (Chen et al., 2009 (link)) and incubated in PBS with or without 50 μg/ml monoclonal IgD from human myeloma (Athens Research and Technology), 2.5 μg/ml galectin-9 (R&D Systems), or 50 μg/ml IgD premixed with 2.5 μg/ml galectin-9 on ice for 15 minutes. Following washing, basophils were sequentially stained with F(ab)2 to IgD biotin (Southern Biotech) and streptavidin (Life Technologies) along with antiFcɛRI FITC (clone: AER-37; eBioscience) and anti-CD44 PE (clone: IM7; Biolegend). Following additional washing, stained cells were fixed with 1 % paraformaldehyde, stained with 100 ng/ml DAPI (Sigma-Aldrich), and imaged. A similar strategy was used to image basophils included in splenocytes from Balb/c mice. These cells were incubated with an Fc-blocking 2.4G2 mAb (Tonbo Bioscience) and subsequently stained with anti-FcɛRI FITC (clone: MAR-1), anti-IgD PE (clone: 11–26c.2a) (BD Biosciences), anti-CD49b APC (clone: DX5) (Biolegend), and Ghost Violet 510 viability dye (Tonbo Bioscience). Following washing, stained cells were fixed with 1 % paraformaldehyde and stained with 5 μg/ml 7-AAD (Tonbo Bioscience).
Galectin 9
Manufactured by R&D Systems
Sourced in United States
Galectin-9 is a carbohydrate-binding protein that belongs to the galectin family. It plays a role in various biological processes, including immune regulation, cell adhesion, and apoptosis. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant mouse galectin 9, by R&D Systems (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Dectin 1 igg fc, by InvivoGen (2 mentions)
Pngase f, by InvivoGen (2 mentions)
Dectin 1 igg fc, by R&D Systems (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Galectin 1, by R&D Systems (1 mentions)
Mts substrate, by Promega (1 mentions)
Recombinant galectin 1, by R&D Systems (1 mentions)
M5 plate reader, by Molecular Devices (1 mentions)
Super signal pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Spectramax, by Molecular Devices (1 mentions)
Streptavidin, by Thermo Fisher Scientific (1 mentions)
Anti cd44 pe, by BioLegend (1 mentions)
Anti cd49b apc, by BioLegend (1 mentions)
Ghost violet 510 viability dye, by Cytek Biosciences (1 mentions)
Anti igd pe, by BD (1 mentions)
7 aad, by Cytek Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
9 protocols using galectin 9
1
Basophil IgD and Galectin-9 Interactions
2
Dectin-1 IgG Fc Glycosylation Profiling
3
Plasma Galectin Levels in Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentration of gal-1, gal-8 and gal-9 was measured in plasma of healthy donors (n = 160) and cancer patients (n = 160) (see Supplementary Fig. 3 ). For ELISA testing, plasma samples were diluted 3-fold for galectin-1 (R&D Systems) and galectin-8 (Ray Biotech), and 10-fold for galectin-9 (R&D Systems). All ELISAs were performed according to the manufacturer’s recommendations.
4
Dectin-1 IgG Fc Glycosylation Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dectin-1 IgG Fc (8 μg; InvivoGen) was treated with PNGase F (20 μl, ~10,000 units; New England Biolabs) following the company’s non-denaturing protocol (37°C, 24 hr). PNGase F treated and untreated Dectin-1 IgG Fc samples (4 μg; InvivoGen) were added to protein G beads (25 μl, Dynabeads Protein G; novex) and the mixtures were incubated at room temperature for 10 min. The beads were washed 2 × with PBS-T (pH 7.4 with 0.02% tween). Recombinant mouse Galectin-9 (4 μg; R&D Systems) was resuspended in PBS-T or a solution of 100 mM lactose in PBS-T. Galectin-9 samples were then incubated with the Dynabead/Dectin-1 IgG Fc complex for 20 min at room temperature. The Dynabeads were then washed 3 × with PBS-T and transferred to a clean tube. Galectin-9 and Dectin-1 IgG Fc were eluted in SDS-PAGE buffer (PBS with 10% BME) by heating at 98°C for 10 min. The samples were then analyzed by SDS-PAGE and stained with Coomassie Blue.
5
IgD Binding to Human Basophils
6
Galectin-9 Modulates Jurkat and KE-37 Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The influence of galectin-9 (R&D Systems, USA) on the proliferation of Jurkat and KE-37 cell lines was measured using 3-(4,5-dimethylthiazol-2-yl) -5-(3- carboxymethoxy-phenyl)-2- (4-sulfophenyl)- 2H-tetrazolium (MTS) cell viability assay (20 ). For this experiment, Jurkat (3 × 103 cells/well) and KE-37 (3 × 103 cells/well) cell lines were plated in 96-well plates and incubated with galectin-9 at 0.1-100 nM. After 48 h of growth, 20 μL of the MTS substrate (Promega, Tokyo, Japan) was added to each well and then were incubated for an additional 3 h. Then, the absorbance was read at 490 nm using a microplate reading spectrophotometer (BioTek, USA). According to the documents obtained from the MTS assay, the IC50 of galectin-9 for Jurkat and KE-37 cell lines was calculated. The IC50 values were determined for 0.1-100 nM concentration of galectin-9 using the GraphPad Prism software.
7
Recombinant Galectin Production and Probes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant galectin-1 and galectin-9 were in-house produced or purchased from R&D systems. For fluorescent anisotropy and apoptosis assays, the oxidation resistant mutant galectin-152 (link), i.e., galectin-1 C3S (in-house produced) was used.
The synthetic high-affinity tdga-probe (0.1 µM, 3′-[4-(fluorescein-5-amidomethyl)−1H-1,2,3-triazol-1-yl]−3′-deoxy-β-D-galactopyranosyl 3-(3,5-dimethoxybenzamido)−3-deoxy-1-thio-β-D-galactopyranoside)9 (link) as well as the low affinity probe (lacto-N-tetraose; LNT-probe)53 (link) were in-house produced.
Chemokines CXCL4 and CCL5 were either in-house produced51 (link) or purchased from R&D systems. All other chemokines and cytokines were purchased from R&D systems. The beta-sheet region, CXCL422-54, was chemically synthesized and purchased from Schafer-N. Anginex was a kind gift from Prof. K. Mayo (University of Minnesota, Minneapolis, USA). Antibodies were purchased from BD Biosciences unless indicated otherwise.
The synthetic high-affinity tdga-probe (0.1 µM, 3′-[4-(fluorescein-5-amidomethyl)−1H-1,2,3-triazol-1-yl]−3′-deoxy-β-D-galactopyranosyl 3-(3,5-dimethoxybenzamido)−3-deoxy-1-thio-β-D-galactopyranoside)9 (link) as well as the low affinity probe (lacto-N-tetraose; LNT-probe)53 (link) were in-house produced.
Chemokines CXCL4 and CCL5 were either in-house produced51 (link) or purchased from R&D systems. All other chemokines and cytokines were purchased from R&D systems. The beta-sheet region, CXCL422-54, was chemically synthesized and purchased from Schafer-N. Anginex was a kind gift from Prof. K. Mayo (University of Minnesota, Minneapolis, USA). Antibodies were purchased from BD Biosciences unless indicated otherwise.
8
TIM3/Galectin-9 Binding Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 4
Anti-TIM3 variants were tested for their ability to block a TIM3/Galectin9 interaction. Galectin-9 (R&D Systems) was adsorbed on Nunc 384-well white Maxisorp plates at 2 μg/mL in sodium bicarbonate buffer (pH 8.9) and incubated at 30° C. for 1 hour or overnight at 4° C. The plate was washed 3 times with PBS pH 7.4 with 0.05% Tween20 and blocked with 2% bovine serum albumin (BSA) in PBS pH 7.4+0.1% Tween20 for 1 hour at 30° C. The blocking solution was aspirated, and a dilution series of antibody was mixed with 10 nM biotinylated TIM3-Fc (R&D Systems) in 0.2% BSA in PBS pH 7.4+0.1% Tween20 (diluent buffer) and incubated at 30° C. for 1 hour. The plate was washed, and streptavidin-HRP (Pierce) in diluent buffer was added to all wells. After 1 hour incubation at 30° C., the plate was washed, followed by detection with SuperSignal Pico Chemiluminescent Substrate (Thermo Pierce). Luminescence was detected on a SpectraMax® M5 plate reader (Molecular Devices).
9
Western Blot Analysis of Exosomal Galectins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated exosome samples (20 µl/sample; 8-10 µg exosomes, aroud 40 µg lysate and 20 pg recombinant galectins) were mixed with Laemmli sample buffer (Bio-rad) containing 50 mM dithiothreitol (DTT) and incubated at 95 °C for 5 minutes to reduce and denaturate proteins before loading into a 4-20% SDS-PAGE gel (Mini-PROTEAN® Bio-Rad, Veenendaal, The Netherlands) for separation by electrophoresis. Separated proteins were then transferred into a polyvinylidene difluoride membrane (Transblot Turbo, Bio-Rad) after which the membrane was blocked with 5% milk protein in phosphate-buffered saline containing 0.05% Tween-20. The membranes were then incubated with galectin-4 (1:4000) and galectin-9 (1:100) antibodies (both from R&D). After overnight incubation, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000, Dako, Santa Clara, CA, USA) for 2 h. After washing, ECL reagent (Cytiva, Marlborough, MA, USA) was used to visualize the proteins. Image J software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) was used to analyze the data.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!