Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors for measurement of proliferative capacity in the presence of recombinant human galectin-9 (R&D Systems, Minneapolis, MN). In three separate experiments, cells from two allogeneic healthy controls were mixed together: one of the PBMC samples having been irradiated (stimulator). Culture conditions included 20U/mL IL-2 in RPMI + 10% fetal bovine serum (FBS) and experimental wells received 2ug/mL galectin-9. In a 96-well plate, 100,000 responder cells in 100uL were added with 100uL of serial diluted irradiated cells in triplicate. Cultures were incubated for 3 days at 37°C and 5% CO2 before addition of 1uC/well of H3 thymidine overnight. The plate was washed and transferred to a 96-well filter plate. Scintillation fluid was added and counts per minute (cpm) were recorded on a scintillation counter. Averages and standard deviations were calculated from each replicate and combined between all three experiments.
Recombinant human galectin 9
Manufactured by R&D Systems
Sourced in United States
Recombinant human galectin-9 is a soluble protein produced in E. coli cells. Galectin-9 is a member of the galectin family of carbohydrate-binding proteins.
Automatically generated - may contain errors
3 protocols using recombinant human galectin 9
1
Galectin-9 Induced PBMC Proliferation
2
Galectin-9 Binding Assay with TIM-3 Fc
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Recombinant human galectin-9 (R&D Systems) was conjugated to PE using the Lightning Link R-PE Antibody Labeling Kit (novusbio) and TIM-3 Fc (R&D Systems) was coupled to microspheres using Luminex xMAP Antibody Coupling Kit (Luminex). On the day of the competition assay, 60 μl of 5 × 106 2 μg/ml Luminex TIM-3 Fc beads were diluted in 2.94 ml Assay Buffer (Millipore). 25 μl/well of diluted beads were added to a non-binding mylar 96-well plate (Grienier). 25 μl/well of antibodies were serially diluted in Assay Buffer. Plates were incubated for 1.5 hours at RT on a shaker, washed, and human galectin-9 PE at a concentration of 4 μg/ml was added. Plates were incubated for 45 minutes at RT on a shaker, washed, and 9 μl/well of Sheath Fluid (Luminex) was added to the wells. Plates were read on a FlexMap 3D.
3
Analyzing Galectin-9 and TIM-3 in Inflammation
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Recombinant human Galectin-9 and anti-human TIM-3 antibody were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human SAA was purchased from Peprotech (Rocky Hills, NJ). Antipro-IL-β Polyclonal antibody (MBS 125139) was purchased from MyBioSource (San Diego, CA USA). Anticleaved-IL-β (p17, D3A3Z) antibody was purchased from Cell Signaling Technology (CST, Danvers, USA).
Anti-NLRP-3 antibody was purchased from MERCK MILLIPORE (Billerica,MA USA).
Anti-NLRP-3 antibody was purchased from MERCK MILLIPORE (Billerica,MA USA).
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