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 is a powerful tool for cell labeling, tracking and proteomics analysis. However, the effects of metabolic labeling agents on cell metabolism and physiology are still unclear. To address this issue, we investigated the effects of Ac4ManNAz treatment on cells using microarray analysis and analysis of membrane channel activity, individual biophysiological characteristics and glycolytic flux. The results showed that 50 μM Ac4ManNAz treatment led to a reduction in major cellular functions, including energy production capacity, cell infiltration capacity and channel activity. Interestingly, 10 μM Ac4ManNAz had the least effect on the cellular system and had sufficient labeling efficiency for cell labeling, tracking and proteomics analysis. Based on our results, we recommend 10 μM as the optimal concentration of Ac4ManNAz for in vivo cell labeling and tracking. Furthermore, we anticipate that this method can be used for cell-based therapy to monitor the efficiency of molecular delivery and the fate of recipient cells. [1] Metabolic labeling is one of the most effective methods for tracking live cells in vitro and in vivo. However, the mechanisms by which metabolic agents affect cells through modification of glycosylation have not been fully elucidated. Therefore, metabolic labeling has not been widely used for tracking and labeling endothelial progenitor cells (EPCs). This study analyzed the cellular functional properties (such as proliferation, migration and permeability) and gene expression patterns of human umbilical cord blood endothelial progenitor cells (hUCB-EPC) treated with metabolic labeling reagents to elucidate the cellular effects of metabolic labeling reagents. The results showed that 10 μM Ac4ManNAz treatment had no effect on cell function or gene regulation, but higher concentrations of Ac4ManNAz (>20 μM) led to inhibition of cellular functional properties (proliferation rate, cell viability and endocytosis) and downregulation of gene expression associated with cell adhesion, PI3K/AKT, FGF and EGFR signaling pathways. Interestingly, the angiogenesis and angiogenic potential of hUCB-EPC were not affected by the concentration of Ac4ManNAz. Based on our results, we suggest that 10 μM is the optimal concentration of Ac4ManNAz for labeling and tracking hUCB-EPC in vivo. In addition, we expect our method to be used to understand the efficacy and safety of stem cell therapy in vivo. [2]

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Sialidized glycans are elevated in a variety of cancers and are associated with disease progression. However, specific glycoproteins involved in sialylation on the surface of cancer cells have not been adequately characterized, especially in real human disease tissues. Metabolic and bioorthogonal labeling methods have previously been used to enrich and identify sialic acid glycoproteins from cultured cells and model organisms. In this paper, we report for the first time the application of this glycoproteomics platform to cultured human tissues in vitro. Normal and cancerous prostate tissue sections were cultured in the presence of Ac4 ManNAz, an azide-functionalized sialic acid biosynthetic precursor. This compound is metabolized to azidesialic acid and integrated into sialic acid glycoproteins on the cell surface and in secretion. Glycoproteins that were elevated or specific to cancerous prostate tissues were identified by chemibiotinylation, enrichment, and mass spectrometry. Thus, this work extends the application of bioorthogonal labeling strategies to clinically significant problems. [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.)