glycoprotein labeling reagent

For cell labeling, tracking, and proteome analysis, Ac4ManNAz (10 μM) has enough labeling efficiency with little effect on biological systems [1]. Major cellular processes such as energy production, cell infiltration, and channel activity are all reduced by Ac4ManNAz (50 μM) [1].

It is suggested that 10 μM should be used as the optimal concentration of Ac4ManNAz for in vivo cell labeling and tracking of hUCB-EPCs. Additionally, we expect that our approach can be used for understanding the efficacy and safety of stem cell-based therapy in vivo and to help determine the utility of stem cells in downstream experiments.[2]

Mitochondrial membrane potential was measured using JC-1 dye (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) according to the manufacturer's instructions. Briefly, Ac4MAnNAz-treated or untreated cells were incubated with 10 µg/mL JC-1 dye for 15 min, and fluorescence images were taken using a 20x objective. The ratio of red fluorescence JC-1 aggregates and green JC-1 monomers was measured using image J following image background correction.[1]

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Measurement of labeled protein using 10 μM Ac4ManNAz [1]
The A549 cells were grown with 0 and 10 μM Ac4ManNAz in a 6-well plate. Before analysis, cells were washed with PBS and harvested. Before the protein isolation, the cells were mixed so that the ratio gradually increased to 100% of 10 μM Ac4ManNAz-treated cells. Samples each had a total of 5×105 cells in 1 mL. Mixed samples were lysed in whole-cell lysis buffer (10% glycerol, 0.5 mM EDTA, 1 mM DTT, 2 mM sodium fluoride, 0.2% Triton X-100 in PBS pH 7.4) supplemented with protease and phosphatase inhibitors. The total proteins were incubated with DBCO-Cy5 (20 μM, final concentration) for 1 h at 37 °C and precipitated using ethanol. The precipitated proteins were resuspended in PBS and analyzed using a fluorescence microplate reader at 588 nm. The data shown are the average of three separate experiments, each performed in triplicate.

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Analysis of reactive oxygen species (ROS) generation and mitochondrial membrane potential [2]
Microscopic fluorescence imaging was used to study reactive oxygen species (ROS) generation in hUCB-EPCs after treatments to different concentrations of Ac4ManNAz. Cells (1 × 104 per well) were seeded and were then treated to 0 µM, 10 µM, 20 µM and 50 µM concentrations of Ac4ManNAz for 3 days at 37 °C. Cells were incubated with 2,7-Dichlorodihydrofluorescein diacetate (DCF-DA) (10 mM) for 30 min at 37 °C. The reaction mixture was aspirated and replaced by 200 µl of phosphate-buffered saline (PBS) in each well. The plate was kept on a shaker for 10 min at room temperature in the dark. An inverted fluorescent microscope was used to visualize intracellular fluorescence of cells and to capture images. Mitochondrial membrane potential was measured using JC-1 dye (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) according to the manufacturer’s instructions. Briefly, hUCB-EPCs were grown in 24-well plate and treated with different concentrations of Ac4ManNAz. Ac4MAnNAz-treated or untreated hUCB-EPCs were washed with PBS and stained with 10 µg/mL JC-1 dye for 15 min at 37 °C and fluorescence images were acquired.

Invasion and wound healing
Matrigel (100 μL; 7-8 mg/mL) in serum-free medium was added to each well of a Transwell Corning Costar plate and dried overnight in a culture hood. The following day, 2.5 × 104 cells in serum-free medium were pipetted onto the Matrigel, and complete medium was added to the bottom chamber. Following incubation, the transmembrane filter was stained with crystal violet and the number of cells counted. For wound healing, a small area was cleared along the diameter of the 10 cm dishes through confluent monolayers of A549 and Az4MAnNAz-treated A549 cells using a sterile pipette tip. Cell migration was measured and photographed from the wound/scratch edge every 8 h.[1]

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In vitro cell labeling and imaging [2]
hUCB-EPCs (5 × 104 cells/35 mm glass-bottom dishes) were treated with Ac4ManNAz, Ac4GalNAz, or Ac4GlcNAz supplemented medium (50 µM, final concentration of each) for 72 h. Cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS) and subsequently incubated with DBCO-Cy5 (10 µM, final concentration) for 1 h at 37 °C. Cells were then washed and fixed with 4% paraformaldehyde for 15 min. After fixation, nuclei were stained with DAPI solution

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Cell viability and wound healing assay [2]
To measure cell viability, hUCB-EPCs were seeded in 96-well plates (5 × 103 cells/well) and incubated for 1 day. Cells were incubated with various concentrations of Ac4MAnNAz (0 to 50 µM) for 3 days at 37 °C. Cell Counting Kit-8 solution (10 µL) was then added to each well. After further incubation for 2 h at 37 °C, the absorbance of each well was measured at 450 nm using a microplate reader. For wound healing, a sterile pipette tip was used to clear a small area across the diameter of 10 cm dishes with confluent monolayers of untreated or Ac4MAnNAz-treated hUCB-EPCs. Cell migration was measured and photographed from the wound/scratch edge after 18 h.

[1]. Physiological Effects of Ac4ManNAz and Optimization of Metabolic Labeling for Cell Tracking. Theranostics. 2017 Mar 1;7(5):1164-1176.

[2]. Safety and Optimization of Metabolic Labeling of Endothelial Progenitor Cells for Tracking. Sci Rep. 2018 Sep 4;8(1):13212.

[3]. Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics. Angew Chem Int Ed Engl. 2017 Jul 24;56(31):8992-8997.

Metabolic labeling techniques are powerful tools for cell labeling, tracking and proteomic analysis. However, at present, the effects of the metabolic labeling agents on cell metabolism and physiology are not known. To address this question, in this study, we analyzed the effects of cells treated with Ac4ManNAz through microarray analysis and analyses of membrane channel activity, individual bio-physiological properties, and glycolytic flux. According to the results, treatment with 50 μM Ac4ManNAz led to the reduction of major cellular functions, including energy generation capacity, cellular infiltration ability and channel activity. Interestingly, 10 μM Ac4ManNAz showed the least effect on cellular systems and had a sufficient labeling efficiency for cell labeling, tracking and proteomic analysis. Based on our results, we suggest 10 μM as the optimum concentration of Ac4ManNAz for in vivo cell labeling and tracking. Additionally, we expect that our approach could be used for cell-based therapy for monitoring the efficacy of molecule delivery and the fate of recipient cells. [1]

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Metabolic labeling is one of the most powerful methods to label the live cell for in vitro and in vivo tracking. However, the cellular mechanisms by modified glycosylation due to metabolic agents are not fully understood. Therefore, metabolic labeling has not yet been widely used in EPC tracking and labeling. In this study, cell functional properties such as proliferation, migration and permeability and gene expression patterns of metabolic labeling agent-treated hUCB-EPCs were analyzed to demonstrate cellular effects of metabolic labeling agents. As the results, 10 μM Ac4ManNAz treatment had no effects on cellular function or gene regulations, however, higher concentration of Ac4ManNAz (>20 μM) led to the inhibition of functional properties (proliferation rate, viability and rate of endocytosis) and down-regulation of genes related to cell adhesion, PI3K/AKT, FGF and EGFR signaling pathways. Interestingly, the new blood vessel formation and angiogenic potential of hUCB-EPCs were not affected by Ac4ManNAz concentration. Based on our results, we suggest 10 μM as the optimal concentration of Ac4ManNAz for in vivo hUCB-EPC labeling and tracking. Additionally, we expect that our approach can be used for understanding the efficacy and safety of stem cell-based therapy in vivo. [2]

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Sialylated glycans are found at elevated levels in many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to the cancer cell-surface sialylation are not well characterized, specifically in bona fide human disease tissue. Metabolic and bioorthogonal labeling methods have previously enabled the enrichment and identification of sialoglycoproteins from cultured cells and model organisms. Herein, we report the first application of this glycoproteomic platform to human tissues cultured ex vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of the azide-functionalized sialic acid biosynthetic precursor Ac4 ManNAz. The compound was metabolized to the azidosialic acid and incorporated into cell surface and secreted sialoglycoproteins. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance. [3]

C16H22N4O10

430.366684436798

430.133

C, 44.65; H, 5.15; N, 13.02; O, 37.17

1213701-11-1

Ac4ManNAz;361154-30-5

98290519

White to off-white solid powder

1.2

1

12

12

30

736

5

CC(=O)OC[C@@H]1[C@H]([C@@H]([C@@H]([C@H](O1)OC(=O)C)NC(=O)CN=[N+]=[N-])OC(=O)C)OC(=O)C

HGMISDAXLUIXKM-LIADDWGISA-N

InChI=1S/C16H22N4O10/c1-7(21)26-6-11-14(27-8(2)22)15(28-9(3)23)13(16(30-11)29-10(4)24)19-12(25)5-18-20-17/h11,13-16H,5-6H2,1-4H3,(H,19,25)/t11-,13+,14-,15-,16?/m1/s1

Peracetylated N-azidoacetyl-d-mannosamine

(alpha)-Ac4ManNAz; 1213701-11-1; (2R,3S,4R,5S,6R)-6-(Acetoxymethyl)-3-(2-azidoacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate; (; A)-Ac4ManNAz;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (232.36 mM)

≥ 2.50 mg/mL (5.80 mM) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline
≥ 2.50 mg/mL (5.80 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.50 mg/mL (5.80 mM) in 10% DMSO + 90% Corn oil  (Please use freshly prepared in vivo formulations for optimal results.)