ac 4 mannaz  (MedChemExpress)

Journal: bioRxiv

Article Title: Enzyme-Linked Cycloaddition Assay (ELCA) for rapid, ultra-sensitive monitoring of secreted sialoglycoproteins

doi: 10.64898/2026.06.15.732414

Figure Lengend Snippet: A-B) Lectin-based flow cytometry analysis of cell surface glycoproteins using MAL-II (A; Neu5Acα2-3Galβ1-3GalNAc) and PNA (B; Galβ1-3GalNAc) lectin staining following inhibition of translation with 25 µM cycloheximide (CHX) for 24 h showed no significant changes (n = 3). C) Under the same treatment conditions, a clear reduction in newly secreted sialoglycoproteins was detected using ELCA. Measurements were performed under serum-free conditions using standard colorimetric detection (n = 8). D-E) A375 cells were metabolically labeled with 50 µM Ac 4 ManNAz for 72 h, followed by re-seeding in fresh serum-containing DMEM (D) or serum-free Opti-MEM (E) medium. Supernatant samples were collected at the indicated time-points and assayed with ELCA using high-sensitivity luminescent detection (n = 4). F-G) Serum-containing DMEM medium supplemented with Ac 4 ManNAz (F: 50 µM; G: 25 µM; H: 5 µM) was added to A375 cells. At the indicated time points, the cells were then incubated in fresh serum-free Opti-MEM for 1 h, after which supernatants containing proteins secreted during this period were collected and stored at −20 °C for ELCA analysis using high-sensitivity luminescent detection (n = 4). Glycan structures have been drawn with GlycoDraw and conform with the Symbol Nomenclature For Glycans (SNFG). All assays were conducted in A375 cells. Statistical significance was assessed using a two-tailed Welch’s t-test (two conditions) or one-way ANOVA with Dunnett’s multiple comparisons test (>2 conditions). *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Unless stated otherwise, metabolic labeling was performed by growing the cells in the presence of 50 μM Ac 4 ManNAz (CLK-1084-25, Jena Bioscience) for 72 h. Small molecule-mediated modulation of cellular glycosylation was performed by treatments with 1 mM Benzyl-α-GalNAc (HY-129389, MedChemExpress) and 10 μM Kifunensine (HY-19332, MedChemExpress).

Techniques: Flow Cytometry, Analysis, Staining, Inhibition, Metabolic Labelling, Labeling, Sensitivity, Incubation, Glycoproteomics, Two Tailed Test

Journal: bioRxiv

Article Title: Enzyme-Linked Cycloaddition Assay (ELCA) for rapid, ultra-sensitive monitoring of secreted sialoglycoproteins

doi: 10.64898/2026.06.15.732414

Figure Lengend Snippet: A) Overview of the experimental setup. A375 cells were metabolically labeled with 50 µM Ac 4 ManNAz for 72 h, followed by re-seeding in fresh serum-containing medium supplemented with common glycosylation inhibitors (1 mM Benzyl-α-GalNAc, Benz, and 10 µM Kifunensine, Kif). At the indicated time points, supernatants were collected and stored at −20 °C until ELCA analysis. Cells were washed once with Opti-MEM, then incubated with 10 µM DBCO-biotin for 1 h at 37 °C and subsequently analyzed by flow cytometry. B) Flow cytometry analysis of biotinylated cell surface glycoproteins using fluorescein-conjugated streptavidin for detection (n = 4). C) ELCA measurements of serum-containing supernatants using high-sensitivity luminescent detection (n = 4), reflecting cumulative protein secretion for the duration of the treatment. All assays were conducted in A375 cells. Ctrl: untreated control. Statistical significance was assessed using two-way ANOVA with Tukey's multiple comparisons test (<2 conditions, 2 variables). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Unless stated otherwise, metabolic labeling was performed by growing the cells in the presence of 50 μM Ac 4 ManNAz (CLK-1084-25, Jena Bioscience) for 72 h. Small molecule-mediated modulation of cellular glycosylation was performed by treatments with 1 mM Benzyl-α-GalNAc (HY-129389, MedChemExpress) and 10 μM Kifunensine (HY-19332, MedChemExpress).

Techniques: Metabolic Labelling, Labeling, Glycoproteomics, Analysis, Incubation, Flow Cytometry, Sensitivity, Control, FACS, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Enzyme-Linked Cycloaddition Assay (ELCA) for rapid, ultra-sensitive monitoring of secreted sialoglycoproteins

doi: 10.64898/2026.06.15.732414

Figure Lengend Snippet: A) Overview of the experimental setup. THP-1 cells were differentiated into naïve macrophage-like (M0) cells and metabolically labeled (20 nM PMA, 50 µM Ac 4 ManNAz for 72 h), followed by 24 hours of rest and subsequent activation with LPS (100 ng/mL, M1) or IL-4 and IL-13 (20 ng/mL each, M2). At the indicated time points, serum-containing supernatants were collected and stored at −20 °C until ELCA analysis. Cells were then incubated in fresh serum-free Opti-MEM for 1 h, after which supernatants containing proteins secreted during this period were collected and stored at −20 °C for ELCA analysis. B) ELCA measurements of serum-containing supernatants using high-sensitivity luminescent detection (n = 4), reflecting cumulative protein secretion for the duration of the treatment. At each timepoint, letters indicate statistical groupings. C-E) ELCA measurements of serum-free supernatants using high-sensitivity luminescent detection (n = 4), reflecting the abundance of proteins secreted during the 1 h incubation period. All assays were conducted in THP-1 cells. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons test (>2 conditions, 1 variable) or two-way ANOVA with Tukey’s multiple comparisons test (>2 conditions, 2 variables). **p < 0.01, ****p < 0.0001.

Article Snippet: Unless stated otherwise, metabolic labeling was performed by growing the cells in the presence of 50 μM Ac 4 ManNAz (CLK-1084-25, Jena Bioscience) for 72 h. Small molecule-mediated modulation of cellular glycosylation was performed by treatments with 1 mM Benzyl-α-GalNAc (HY-129389, MedChemExpress) and 10 μM Kifunensine (HY-19332, MedChemExpress).

Techniques: Metabolic Labelling, Labeling, Activation Assay, Analysis, Incubation, Sensitivity

Journal: EJNMMI Radiopharmacy and Chemistry

Article Title: Aluminum-[ 18 F]fluoride radiolabeling of triarylphosphines for cell labeling via the perfluoroaryl azide Staudinger ligation

doi: 10.1186/s41181-025-00409-9

Figure Lengend Snippet: A . In vitro cytotoxicity of compound 1 in human Jurkat T lymphoblasts after 24, 48, and 72 h incubation at 10–250 μM. Negative (–) and positive ( +) controls were complete medium with 0.5% v/v DMSO and lysis Buffer (1:10 v/v), respectively. Columns represent mean ± s.d. (n = 4). The statistical significance was analyzed using unpaired Student’s t -test versus the negative control (* p < 0.05, ** p < 0.01, *** p < 0.001, and n.s. = not significant). The dashed pink line indicates 80% cell viability, the threshold for acceptable cytocompatibility. B – D . Flow cytometry analysis of live Jurkat cells after a 72 h treatment with 1 × PBS (dotted line in B and dashed line in C ) or MGE with Ac 4 MAnNAz (solid line in B – D ) or 1 (complex line in D ), followed by incubation with 1 × PBS (dotted line in B ) or 1 µM phosphine-PEG3-biotin (solid line in B – D and complex line in D ) and finally incubation with streptavidin-FITC (all lines)

Article Snippet: Jurkat cells (5 × 10 5 cells in 1.5-mL complete medium per well) were seeded in a non-treated flat-bottom polystyrene 6-well plate (Avantor, Radnor, PA, USA) and incubated in triplicates (n = 3) with 50 μM of 1 , 50 μM of Ac 4 ManNAz (Lumiprobe, Westminster, MD, USA) or 0.5% (v/v) DMSO in complete medium for 72 h in a cell incubator set at 37 oC with 5% CO 2 and 95% relative humidity.

Techniques: In Vitro, Incubation, Lysis, Negative Control, Flow Cytometry

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: GlycoRNA-L and glycoRNA-S mediate human monocyte adhesion via binding to Siglec-5

doi: 10.1016/j.bbamcr.2025.120017

Figure Lengend Snippet: Expression of glycoRNA-L and glycoRNA-S in human monocytes. (A) THP1 cells were treated with or without Ac 4 ManNAz. RNAs were extracted from the cells and treated with or without RNase. Then the RNA samples reacted with DBCO-PEG4-biotin. RNAs were analyzed on an agarose gel (left) and then the blot was probed with an anti-biotin antibody (right). Representative images are shown. (B) 10 μg of biotinylated total RNAs from THP1 cells were incubated with 1 μl of RNase cocktail (containing RNase A 0.5 U/μ/ and RNase T 20 U/μl), DNase I (1 U/μl), PNGase F (1 U/μl) or Proteinase K (2 μg/μl) at 37 °C for 10 min. After cleaning up by zymo column, the RNA samples were analyzed by northern blot as above. (C) Blotting of cellular or in vitro Ac 4 ManNAz-labeled RNA. Cells were treated with 100 μM Ac 4 ManNAz for 24 h while native RNA in vitro was treated with 0, 5, 10 and 20 mM of Ac4ManNAz at 37 °C for 2 h. (D) THP1 cells were treated with Ac 4 ManNAz and different doses of NIG-1 or kifunensine as indicated. RNAs were extracted from the cells and analyzed by northern blot. Representative images are shown. (E) RNA from different cells treated with 100 μM Ac 4 ManNAz for 24 h and then analyzed by the same procedure as above. Representative images are shown. (F) RNA from THP1 or hPBMC (ATCC) treated with 100 μM Ac 4 ManNAz plus or not plus LPS (1 μg/ml) for 24 h and then analyzed by the same procedure as above. Representative images are shown.

Article Snippet: Stocks of N -azidoacetylmannosamine-tetraacylated (Ac 4 ManNAz or ManNAz, Tocris Bioscience, Cat#:7479) were made to 500 mM in sterile dimethyl sulfoxide (DMSO).

Techniques: Expressing, Agarose Gel Electrophoresis, Incubation, Northern Blot, In Vitro, Labeling

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: GlycoRNA-L and glycoRNA-S mediate human monocyte adhesion via binding to Siglec-5

doi: 10.1016/j.bbamcr.2025.120017

Figure Lengend Snippet: Characterization of structures of glycoRNA-L and glycoRNA-S in THP1 cells. (A) THP1 cells were labeled by 100 μM of Ac 4 ManNAz (ManNAz), GalNAz, GlcNAz and FucAz for 24 h respectively. RNAs were extracted from the cells. Then the RNA samples reacted with DBCO-PEG4-biotin. RNAs were analyzed on an agarose gel (left) and then blotted with an anti-biotin antibody (right). Representative images are shown. (B) Blot of RNA from HeLa cells labeled by same reagents as above. (C) THP1 cells were pretreated with or without NIG-1 (10 μM/L) or kifunensine (5 μM/L) for 1 h and then incubated with GalNAz (100 μM/L) for 24 h. The RNA was isolated and analyzed as described above. (D) 10 μg biotinylated total RNA from Ac 4 ManNAz-labeled THP1 cells were treated with 1 μl RNase cocktail and then directly loaded on the denaturing gel and analyzed by same procedure as above.

Article Snippet: Stocks of N -azidoacetylmannosamine-tetraacylated (Ac 4 ManNAz or ManNAz, Tocris Bioscience, Cat#:7479) were made to 500 mM in sterile dimethyl sulfoxide (DMSO).

Techniques: Labeling, Agarose Gel Electrophoresis, Incubation, Isolation