α lactose

Manufactured by Merck Group

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α-lactose is a disaccharide compound commonly used in laboratory settings. It is composed of one molecule of glucose and one molecule of galactose. α-lactose serves as a basic laboratory reagent and is often utilized in various biochemical and microbiological applications.

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Lactose (233 citations) α-lactose monohydrate (19 citations) α-Lactose-Agarose (4 citations) α-Lactose-Agarose resin (3 citations) α-lactose agarose affinity resin (2 citations) α-lactose-agarose column (2 citations) α-lactose (lactamyl) agarose (2 citations)

14 protocols using α lactose

Modulating Endotoxemia with MSC Variants

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Mice were randomly divided into five experimental groups (n = 6) and received lipopolysaccharide (LPS, 10 mg/kg, i.p.) or adipose derived-MSC (500 ul, i.v.) injection at 0 and 1 h time points: (1): Normal control group: Receiving 500 ul PBS (i.v.) (2). Untreated group: Endotoxemia model was induced by LPS injection (10 mg/kg, i. p.) (Solarbio, Beijing, China), followed by PBS (500 ul) injection without MSCs (3). Unmodified MSC group: After model establishment, mice were lightly anesthetized by inhalation with isoflurane. 10⁶ MSCs diluted in 500 ul PBS were injected through the penile dorsal vein (4). Gal-9 high-expressing group: MSCs were co-cultured with IFN-γ solution (20 ng/ml) (PeproTech, Rocky Hill, United States) for 72 h in advance. When it is time to inject, MSCs were accurately counted at 10⁶ and dissolved in 500 ul PBS for intravenous injection (5). Gal-9 blocking group: 10⁶ MSCs was diluted in 500 ul PBS containing 10.8 mg/ml α-lactose (α-lactose: the specific antagonist of Gal-9, Sigma-Aldrich), and then used for injection.
All mice were sacrificed 24 h later. Spleen were collected for flow cytometry analysis (detailed procedure is shown below). Serum, and half parts of liver, lung, and kidney were frozen in −80°C for further analysis. Half parts of liver, lung, and kidney were immersed in formalin solution for pathological analysis.

Zhao Y., Yu D., Wang H., Jin W., Li X., Hu Y., Qin Y., Kong D., Li G., Ellen A, & Wang H. (2022). Galectin-9 Mediates the Therapeutic Effect of Mesenchymal Stem Cells on Experimental Endotoxemia. Frontiers in Cell and Developmental Biology, 10, 700702.

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Recombinant Gal-9 and Inhibitor Production

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Recombinant Gal-9 (rGal-9) (also known as Gal-9(0)) containing a truncated 2 amino acid inter-domain linker was produced as described before [30 (link)]. The following inhibitors were used: α-lactose (Sigma-Aldrich, St. Louis, MO, USA), zVAD-fmk (R&D Systems, Inc., FMK001, Wiesbaden, Germany), Bapta (CALBIOCHEM, Merck KGaA, Darmstadt, Germany), EDTA (Sigma-Aldrich, Germany), and R406 (Axon MedChem).

Ustyanovska Avtenyuk N., Choukrani G., Ammatuna E., Niki T., Cendrowicz E., Lourens H.J., Huls G., Wiersma V.R, & Bremer E. (2021). Galectin-9 Triggers Neutrophil-Mediated Anticancer Immunity. Biomedicines, 10(1), 66.

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Enzymatic Characterization of β-Galactosidase

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The enzyme β-galactosidase from B. circulans (Biolacta N-5) was kindly provided by Daiwa Kasei (Osaka, Japan), d-xylose (d-glucose) isomerase was purchased from Hampton Research (Aliso Viejo, CA, USA). Eupergit C and Eupergit C 250 L were a gift from Röhm Pharma (Darmstadt, Germany). O-Nitrophenyl-β-d-galactopyranoside (ONPG), α-lactose, d-galactose, d-tagatose, d-glucose, d-fructose, l-cysteine hydrochloride, and carbazole were purchased from Sigma (St. Louis, MO, USA); bicinchoninic acid (BCA reagent) was from Pierce (Rockford, IL, USA); and all other chemicals were of analytical or HPLC grade. The enzymatic kit for glucose determination was from Spinreact S.A. (Girona, Spain). Cheese wheys were kindly supplied by CONAPROLE (Cooperativa Nacional de Productores de Leche, Uruguay).

Torres P, & Batista-Viera F. (2017). Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(2), 284.

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Heterologous Expression of Cellulolytic Enzymes

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Yeast strain Clavispora NRRL Y-50464 obtained from the Agricultural Research Service Patent Culture Collections (Peoria, IL, USA) was used in this study. Cell cultures were maintained and precultured using yeastpeptone (YP) medium containing 10 g yeast extract, 20 g peptone, and 50 g glucose in one liter distilled water. Escherichia coli TOP10 and Pichia expression and transformation kits from Invitrogen (Carlsbad, CA, USA) were applied for gene cloning and selection procedures. An YP medium amended with 5% cellobiose was used for gene expression assays. All oligosaccharides were purchased from Sigma-Aldrich (St. Louis, MO, USA) including cellobiose, cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, laminaritetraose, laminaripentaose, laminarin, α-lactose, lichenan, salicin, and gentiobiose, and metal ions and chemicals KCl, CaCl₂, ZnCl₂, MgCl₂, CuCl₂, CoCl₂, HgCl₂, FeCl₂, FeCl₃, BaCl₂, PbCl₂, LiCl, NiCl₂, MnCl₂, SDS, triton X-100, 2-furaldehyde (furfural), and 5-(hydroxymethyl)-2-furaldehyde (HMF).

Wang X., Liu Z.L., Weber S.A, & Zhang X. (2016). Two New Native β-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast. PLoS ONE, 11(3), e0151293.

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Galectin-9 Granulocyte Modulation

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Total WBCs or isolated granulocytes (3 × 10⁴ cells) were incubated at 37 °C in 200 μL RPMI + 10% FCS for the indicated time points with/without of Gal-9 or Gal-9(S). After incubation, cells were stained by flow cytometry using CD11b-FITC, CD66acde-PE, or Annexin-V-FITC (Immunotools). In assays with conditioned supernatant, supernatants of treated WBCs were harvested and added to freshly isolated granulocytes with/without α-lactose or sucrose (40 mM, Sigma Aldrich, Zwijndrecht, The Netherlands) for 16 h at 37 °C.

Wiersma V.R., Clarke A., Pouwels S.D., Perry E., Abdullah T.M., Kelly C., Soyza A.D., Hutchinson D., Eggleton P, & Bremer E. (2019). Galectin-9 Is a Possible Promoter of Immunopathology in Rheumatoid Arthritis by Activation of Peptidyl Arginine Deiminase 4 (PAD-4) in Granulocytes. International Journal of Molecular Sciences, 20(16), 4046.

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Autophagy Modulation in Restraint Stress

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One hour before the initiation of the physical restraint, the animals received a single intraperitoneal (i.p.) injection of α-lactose (300 mM, 300 μl/20 g body weight; Sigma, Darmstadt, Germany) [28 (link)] or autophagy inhibitor 3-methyladenine (3-MA) (20 mM, 300 μl/20 g body weight, Selleck, Houston, USA) [29 (link)] in 300 μl of sterile saline or 1 μl of solvent control (DMSO) in 300 μl sterile saline for the controls. After physical restraint, mice were sacrificed and blood samples and spleen specimens were collected for further analyses.

Qin A., Zhong T., Zou H., Wan X., Yao B., Zheng X, & Yin D. (2019). Critical role of Tim-3 mediated autophagy in chronic stress induced immunosuppression. Cell & Bioscience, 9, 13.

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Murine CD4+ T Cell Proliferation Assay

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Murine CD4⁺ T cells were purified from spleens in accordance with the instructions of the manufacturer (BD Biosciences). For the proliferation assay, CD4⁺ T cells at a density of 1 × 10⁶ cells per well were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen, Camarillo, CA, USA) and then co-cultured with UC-MSCs (1 × 10⁵ cells per well) or UC-MSCs with 10.8 mg/mL α-lactose (Sigma-Aldrich) for 4 days. Cell division was determined by measuring the CFSE fluorescence intensity by flow cytometry.

Fan J., Tang X., Wang Q., Zhang Z., Wu S., Li W., Liu S., Yao G., Chen H, & Sun L. (2018). Mesenchymal stem cells alleviate experimental autoimmune cholangitis through immunosuppression and cytoprotective function mediated by galectin-9. Stem Cell Research & Therapy, 9, 237.

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Recombinant LGALS9 Protein Production

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Recombinant LGALS9 (rLGALS9) containing a truncated 2 amino acid interdomain linker (also known as Gal-9(0)) and the physiologically occurring short isoform of LGALS9, LGALS9(S)/Gal-9(S), were produced as described previously.²⁰ (link) For confocal analysis, rLGALS9 was conjugated to DyLight® 594 following the manufacturer's protocol (DyLight 594 NHS Ester; Piercenet, Thermo scientific, 46412).
The following inhibitors were used: α-lactose (Sigma Aldrich, L3625), sucrose (Merck Millipore, 107651), Dynasore (Sigma Aldrich, D7693), UCN-01 (Sigma Aldrich, U6508), U0126 (Sigma Aldrich, U120), GW5074 (Sigma Aldrich, G6416), necrostatin-1 (Sigma Aldrich, N9037), and Z-VAD-fmk (Calbiochem, 627610). Chloroquine was from the LC3B Antibody Kit for Autophagy (Life Technologies, L10382). LGALS9 blocking antibody was from GalPharma.

Wiersma V.R., de Bruyn M., Wei Y., van Ginkel R.J., Hirashima M., Niki T., Nishi N., Zhou J., Pouwels S.D., Samplonius D.F., Nijman H.W., Eggleton P., Helfrich W, & Bremer E. (2015). The epithelial polarity regulator LGALS9/galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux. Autophagy, 11(8), 1373-1388.

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Heterologous expression of phytoene desaturases

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LB plus ampicillin (100 µg/mL) was used for cloning, transformation, plasmid purification and pre-culture steps in bacteria. LB is composed by 1% of tryptone, 0.5% of yeast extract and 0.5% of NaCl. For heterologous expression of phytoene desaturases in bacteria, we used the medium ZYM5052^{49 (link)} supplemented with 100 µg/mL ampicillin (Sigma), 0.05% arabinose (Formedium) and 5 µM riboflavin (Sigma). Cells were grown at 37 °C until OD of 0.6, then the culture was switched to 20 °C overnight to allow for protein expression. ZYM5052 is composed by 1% tryptone (Formedium), 0.5% yeast extract (Formedium), 25 mM Na₂HPO₄ (VWR), 25 mM KH₂PO₄ (Fluka), 50 mM NH₄Cl (Normapur), 5 mM Na₂SO₄ (SDS), 0.5% glycerol, 0.05% glucose, 0.2% α-lactose (Sigma), 2 mM MgSO₄ (Prolabo), 50 µM FeCl₃ (Sigma), 20 µM CaCl₂ (Sigma), 10 µM MnCl₂ (Sigma), 10 µM ZnSO₄ (Sigma), 2 µM CoCl₂ (Sigma), 2 µM CuCl₂ (Sigma), 2 µM NiCl₂ (Sigma), 2 µM Na₂MoO₄ (Sigma), 2 µM Na₂SeO₃ (Sigma) and 2 µM H₃BO₃ (Sigma).

Fournié M, & Truan G. (2020). Multiplicity of carotene patterns derives from competition between phytoene desaturase diversification and biological environments. Scientific Reports, 10, 21106.

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Gal-9 Induced Apoptosis Regulation

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FaDu cells (5 × 10⁴/FACS tube) were pre-incubated with/without the pan-caspase inhibitor zVAD-fmk (10 μM) for 1 h, whereupon Gal-9 (50, 100, 150, and 300 nM) with/without α-lactose (40 mM, Sigma-Aldrich) was added for 1–6 h. Cells were then stained with Annexin V-FITC (ImmunoTools) in calcium buffer or anti-CD47-FITC (BioLegend, Clone CC2C6, San Diego, CA, USA) with isotype control.

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