Ac4mannaz

Manufactured by Thermo Fisher Scientific

Sourced in United States

Ac4ManNAz is a chemical compound used as a tool in biomedical research. It serves as a metabolic labeling agent, allowing the detection and visualization of glycans in living cells through the incorporation of azido-modified sialic acids.

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Lab products found in correlation

Trypsin, by Merck Group (1 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions) Dapi solution, by Thermo Fisher Scientific (1 mentions) Confocal laser scanning microscope, by Leica Microsystems (1 mentions) Genechip human genome u133 plus 2, by Thermo Fisher Scientific (1 mentions) Ingenuity pathways analysis software ipa, by Qiagen (1 mentions) Genespring gx, by Agilent Technologies (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Nupage sample loading buffer, by Thermo Fisher Scientific (1 mentions) Mannac, by Merck Group (1 mentions) Facscalibur, by BD (1 mentions)

6 protocols using ac4mannaz

Tracking Glycosylation in MDA-MB-231 Breast Cancer Cells

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MDA-MB-231 (MDA) breast cancer cells were used as a representative cell
line for tracking of glycosylation processes. Cells with passage number of
<20 were used for all experiments. Cells were grown up in DMEM (amended
with 10% FBS, 2mM L-glutamine, 100 U mL⁻¹ Penstrep, and 1% MEM
non-essential amino acids) at 37 °C with a 5% CO2 atmosphere in vented
culture flasks. Cells were passaged using standard cell line splitting
procedures; in brief, upon reaching high confluence (70–80% surface
coverage) cells were detached with 1X Trypsin (Sigma-Aldrich, Cat. No. 59427C)
and transferred to a fresh culture flask. Trypan blue assay was conducted for
all cell counts. L15 medium was amended with 10% FBS, 2mM L-Glutamine, 1% MEM
non-essential amino acids, and 100 U mL⁻¹ Penstrep. Cells in
L15 medium were incubated at 37°C in moist air.
50 mM Ac₄ManNAz (ThermoFisher, Cat. No. C33366) stock
solutions were prepared by dissolving powder Ac₄ManNAz in 100% DMSO
and stored in the dark at −20°C. 10 mg/mL Alexa Fluor 488 stocks
were prepared by dissolving powder Alexa Fluor 488 in 100% DMSO and stored in
the dark at −20°C.

Phelan J., Altharawi A, & Chan K.L. (2020). Tracking glycosylation in live cells using FTIR spectroscopy. Talanta, 211, 120737.

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Fluorescent Glycocalyx Labeling Protocol

Cited 1 time

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HMECs were supplemented with 50 μM Ac₄ManNAz (Thermo Fisher) in order to incorporate azido groups
into sialic acid residues within the glycocalyx on the cell surface.
The azido groups were subsequently conjugated with Alexa-647-alkyne
(Thermo Fisher) via live-cell compatible copper-catalyzed click chemistry
as previously described.^{33 (link)} Briefly, 2 days
after seeding, the cells were washed three times with cold Dulbecco’s
phosphate buffered saline (DPBS) on ice. The cells were then incubated
with 50 mM CuSO₄, 2.5 mM sodium ascorbate, 1 mM aminoguanidine,
250 mM tris-hydroxypropyltriazolylmethylamine (THPTA) (all Sigma-Aldrich),
and 30 μM AlexaFluor647-alkyne in DPBS without Ca²⁺ and Mg²⁺ for 5 min at 4 °C in the dark. The cells
were then washed three times with cold DPBS before fixation with 4%
paraformaldehyde (Thermo Fisher) in DPBS for 20 min at room temperature.

Almahayni K., Nestola G., Spiekermann M, & Möckl L. (2023). Simple, Economic, and Robust Rail-Based Setup for Super-Resolution Localization Microscopy. The Journal of Physical Chemistry. a, 127(20), 4553-4560.

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Metabolic Labeling and Imaging of hUCB-EPCs

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hUCB-EPCs (5 × 10⁴ cells/35 mm glass-bottom dishes) were treated with Ac4ManNAz, Ac4GalNAz, or Ac4GlcNAz (Invitrogen, Carlsbad, CA, USA) 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 (Invitrogen, Carlsbad, CA, USA). All cell images were obtained using a confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany) equipped with a 405 diode (405 nm) and HeNe-Red (633 nm) lasers.

Han S.S., Shim H.E., Park S.J., Kim B.C., Lee D.E., Chung H.M., Moon S.H, & Kang S.W. (2018). Safety and Optimization of Metabolic Labeling of Endothelial Progenitor Cells for Tracking. Scientific Reports, 8, 13212.

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Transcriptional Profiling of A549 Cells Treated with Ac4ManNAz

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The A549 cells were cultured with 0, 10 and 50 μM Ac4ManNAz (Invitrogen, Carlsbad, CA, USA) and harvested. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the quantity and quality of total RNA was evaluated using a NanoDrop spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA) and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). An Affymetrix GeneChip Human Genome U133 plus 2.0 was used for the microarray experiments according to the manufacturer's instructions (Affymetrix, Santa Clara, CA, USA). For the gene expression array, the data were pre-processed, the cell intensity files (CEL) were generated, and the data were analyzed using GeneSpring GX (v11.0; Agilent Technologies). The data were normalized using a global scale normalization, and differentially expressed genes were selected based on a > 7-fold change. The selected genes were annotated based on NetAffx (http://www.affymetrix.com). Functional enrichment and network analysis was performed using Ingenuity Pathways Analysis software (IPA; Ingenuity Systems, Redwood, CA, USA).

Han S.S., Lee D.E., Shim H.E., Lee S., Jung T., Oh J.H., Lee H.A., Moon S.H., Jeon J., Yoon S., Kim K, & Kang S.W. (2017). Physiological Effects of Ac4ManNAz and Optimization of Metabolic Labeling for Cell Tracking. Theranostics, 7(5), 1164-1176.

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Azido Sugar Labeling of CDCP1

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N2 and ML2 cells were treated with 25 nMazido-modified sugars, tetraacetylated N-azidoacetyl-D- mannosamine (AC₄ManNAz, Invitrogen, Carlsbad, CA), and normal control sugars, N-acetyl-D-mannosamine (ManNAc) for 24 h. Cells were lysed in M-PER lysis buffer with protease inhibitor. After CDCP1 was immunoprecipitated from the indicated amount of cell lystes, click reaction was performed with biotinylated alkyne capture reagent (0.1 mM alkyne biotin, 0.1 mM Tris-triazoleamine catalyst, 1 mMCuSO₄, 2 mM sodium ascorbate) on eluted protein at room temperature for 1 h. Unreactive reagents was removed by chloroform and methanol precipitation followed by solublizing protein in NuPAGE sample loading buffer (Invitrogen, Carlsbad, CA) and analyzed by Western blot with IRDye 800 conjugated streptavidin.

Yang L., Dutta S.M., Troyer D.A., Lin J.B., Lance R.A., Nyalwidhe J.O., Drake R.R, & Semmes O.J. (2015). Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer. Oncotarget, 6(41), 43743-43758.

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Sialic acid labeling with Click-iT® reagent

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To label sialic acid with Click-iT^® metabolic reagent (Invitrogen), cells were pre-incubated with 400 nM Ac4ManNAz (Invitrogen) or negative control substrate 400 nM ManNAc (Sigma-Aldrich) for 24 hours and the cells were labeled with Alexa Fluor488 conjugated alkyne for detection. After incubation for 30 minutes at room temperature, cells were washed and analyzed using FACSCalibur (BD Biosciences). All labeling procedures were performed based on the manufacturer's protocol with modifications as previously described [30 ].

Hsieh C.C., Shyr Y.M., Liao W.Y., Chen T.H., Wang S.E., Lu P.C., Lin P.Y., Chen Y.B., Mao W.Y., Han H.Y., Hsiao M., Yang W.B., Li W.S., Sher Y.P, & Shen C.N. (2016). Elevation of β-galactoside α2,6-sialyltransferase 1 in a fructose-responsive manner promotes pancreatic cancer metastasis. Oncotarget, 8(5), 7691-7709.

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