In the assay, HL-60 cells were grown in RPMI medium, induced with PMA and labeled with 10 mM carboxyfluorescein-succinimidyl ester (CFSE) (Biolegend, UK) at 37 °C for 20 min in the dark and washed twice with PBS containing 10% FBS to remove excessive CFSE. Cells were seeded at 2.5×105 cells/well in 24-well plates and incubated at 37 °C with 5% CO2. At least 24 hr before analysis, 2 μl (1 μM) galectin-9 (Biolegend, UK) was added into each well. One group was treated with our anti-TIM-3 Nanobody (10 µl) as the test group and a standard anti-TIM-3 mAb (10 µl) was added to another group as the positive control. One group of the seeded cells was not treated with galectin-9 as the negative control. At the appropriate point in time, cells were washed twice, re-suspended in PBS buffer, and analyzed immediately using a FACS Calibur flowcytometer (Becton Dickinson, USA).
Galectin 9
Manufactured by BioLegend
Sourced in United States
Galectin-9 is a protein that plays a role in various biological processes, including immune regulation, cell-cell interactions, and cell adhesion. It is a member of the galectin family of lectins, which bind to beta-galactoside carbohydrates. Galectin-9 is expressed in a variety of cell types and tissues, and its functions are an area of active research.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Human th cytokine panel 13 plex, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
Human cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Cellquest pro software, by BD (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Zm241385, by Bio-Techne (1 mentions)
Ctla 4, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Anti cd45 apc, by Thermo Fisher Scientific (1 mentions)
Mrs1754, by Bio-Techne (1 mentions)
Anti cd73 antibody, by Abcam (1 mentions)
Anti cd25 pe, by Thermo Fisher Scientific (1 mentions)
Pe anti human cd28, by Thermo Fisher Scientific (1 mentions)
Cd101, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
5 protocols using galectin 9
1
HL-60 Cells Assay for Galectin-9 and Anti-TIM-3 Nanobody
2
Multiplex Analysis of Plasma Cytokines
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To measure the levels of several cytokines in plasma samples, the Human Th Cytokine Panel 13-plex (IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, and TNF-α; Biolegend, CA, USA) and Human Immune Checkpoint Panel1 12-plex [sCD25 (IL-2Ra), 4-1BB, sCD27, B7.2 (CD86), Free Active TGF-β1, CTLA-4, PD-L1, PD-L2, PD-1, Tim-3, LAG-3, and Galectin-9; Biolegend, USA] were used. Briefly, 25 μl of assay buffer was added into each well. Then, 25 μl of the diluted standard control and plasma samples were added to the wells. The wells were then filled with 25 μl of mixed beads and 25 μl of detection antibodies and incubated for 2 h at room temperature on an orbital plate shaker. Next, 25 μl of streptavidin-PE solution was added, and the plate was shaken orbitally for 30 min at room temperature. Plates were centrifuged at 1,000 rpm for 5 min, liquid was removed, and plates were washed twice with wash buffer. Finally, 150 μl of wash buffer was added to each well, and the plates were shaken for 2–3 min prior to analysis by flow cytometry (BD FACSCalibur™, Becton Dickinson, NJ, USA).
3
Galectin-9 Regulation of CD4+ T Cells
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Purified CD4+T cells isolated from PBMCs of 10 patients by Human CD4+T cell Enrichment kit (Stemcell Technologies, Vancouver, BC, Canada) were cultured in triplicate in a concentration of 4 × 105 cells per well in 200 ul RPMI-1640 containing 10% fetal bovine serum. The cells were stimulated with or without 2 ug/ml galectin-9 (PROSPEC, East Brunswick, USA). The anti-Tim-3 antibodies (5 ug/ml) (Biolegend, San Diego, CA) were added into wells before galectin-9 stimulation or not. The cells not stimulated with galectin-9 or anti-Tim-3 mAb were used as control. Then the cells were harvested after 5 days and stained CD4-PerCP, CD45RA-FITC and Foxp3-APC. After staining, the cells were analyzed with a four-color cytometer (FACS-caliber; CELLQuest Pro software, Beckton Dickinson).
4
Murine T Cell Activation and Characterization
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Anti-mouse CD3 (clone 145-2C11) and anti-CD28 (clone PV-1) antibodies were prepared in our laboratory. DMEM, penicillin and streptomycin from GIBCO Inc. (Grand Island, NY, USA); fetal bovine serum (FBS) from HyClone Inc. (Logan, UT, USA); anti-mouse antibodies including Alexa 488 anti-Foxp3, -IFNγ and -perforin, PE anti-CD25, -CD39, -CD101, -CTLA-4, -granzyme B, ICOS and -IL-2, PerCP-Cy5.5 anti-CD73 and APC anti-CD45.2, FITC anti-human CD8, PE anti-human CD28, APC anti-human CD39 and PE-Cy7 anti-human CD73 from eBioscience (San Diego, CA, USA); PE anti-TNFα, -CCR6, -CD103, -Galectin-9 and -Helios antibodies from BioLegend (San Diego, CA, USA); PE anti-CD122, -CTLA-4, -LAG3 and -PD-1, APC anti-FR4, and PE-Cy7 anti-GITR antibodies from BD Bioscience (San Jose, CA, USA); Anti-CD73 antibody from Abcam (Cambridge, MA, USA); Mitomycin C, LPS, MTT, α, β-methylene adenosine 5’-diphosphate (AMPCP) from Sigma (St. Louis, MO, USA); KC7F2 from Calbiochem (Burlington, MA, USA); ZM241385 and MRS1754 from Tocris (Minneapolis, MN, USA); CFSE from Molecular Probes (Eugene, OR, USA); M2 peptides (LCMV gp33-41, KAVYNFATM) from AnaSpec, Inc. (San Jose, CA, USA); LY294002 from Cell signaling Technology (Danvers, MA, USA) were purchased.
5
Tim-3 Blocking in T2DM PBMC Assay
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For Tim-3 blocking assays, PBMCs from T2DM patients were preincubated with 10 μg/ml of anti-human Tim-3 (BioLegend, Cat#F38-2E2) or Galectin-9 (BioLegend, Cat#9M1-3) in DMEM (Hyclone, USA) containing 10% T2DM serum for 6 h at 37°C. These cells were subsequently stimulated with 50ng/ml IL-12 plus 50ng/ml IL-18 (Proteintech, USA) for evaluating cytokine secretion and granule release.
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