Sta 011

Manufactured by Cell Biolabs

Sourced in United Kingdom, United States

The STA-011 is a laboratory instrument designed for the accurate measurement and analysis of cell viability and proliferation. It utilizes a colorimetric method to determine the number of viable cells in a sample. The STA-011 provides a reliable and efficient way to assess the growth and health of cell cultures in a research or laboratory setting.

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Lab products found in correlation

Lsm 780 nlo, by Zeiss (1 mentions) Ab30917, by Abcam (1 mentions) D0486, by Agilent Technologies (1 mentions) Anti ph2ax, by Merck Group (1 mentions) Anti pgk1, by Proteintech (1 mentions) Anti phospho p53, by Cell Signaling Technology (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Anti β actin, by Merck Group (1 mentions) Anti gapdh, by Proteintech (1 mentions) Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions) P s6k1, by Cell Signaling Technology (1 mentions) Alexa fluor 555 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) T s6k1, by Cell Signaling Technology (1 mentions) Donkey anti rabbit igg fitc, by Santa Cruz Biotechnology (1 mentions) P akt, by Cell Signaling Technology (1 mentions) P mtor, by Cell Signaling Technology (1 mentions) Ab62353, by Abcam (1 mentions) Non fat dried milk, by Bio-Rad (1 mentions) β actin, by Merck Group (1 mentions) Protease and phosphatase inhibitor, by Roche (1 mentions)

5 protocols using sta 011

Quantification of Advanced Glycation End-Products

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Several measurements were performed on transverse slices of skin biopsies. Immunohistochemical staining was conducted with the anti-AGE antibodies anti-CML (ab30917, Abcam, Cambridge, UK) and anti-N^δ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine ([MG-H1], STA-011, cell biolabs, inc., NL) using goat anti-mouse IgG alkaline phosphatase (AP) conjugate ([Go-a-Mo-AP], D0486, Dako, Glostrup, DK) as chromogenic reporter. Histological localization of CML and MG-H1 was evaluated semi-quantitatively by two independent investigators (I.M.A and G.F.H.D). Hematoxylin and eosin (HE) stains were added to be able to identify individual cells.
Invasively assessed intrinsic fluorescence was measured by confocal microscopy using the ZEISS LSM 780 NLO (Zeiss, GER) and quantified by ImageJ. We used single (405nm and 440nm) and 2-photon (750nm, equivalent to 375nm) excitation, which corresponds to the excitation area of the AGE Reader.
In the second skin biopsy, including epidermis and dermis, the concentrations of CML, MG-H1 and pentosidine were assessed by ultra-performance liquid chromatography tandem mass spectrometry measurements (UPLC-MS/MS) and high-performance liquid chromatography (HPLC), respectively [20 , 21 (link), 22 (link)]. (Supplementary material).

Atzeni I.M., Boersema J., Pas H.H., Diercks G.F., Scheijen J.L., Schalkwijk C.G., Mulder D.J., van der Zee P, & Smit A.J. (2020). Is skin autofluorescence (SAF) representative of dermal advanced glycation endproducts (AGEs) in dark skin? A pilot study. Heliyon, 6(11), e05364.

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Western Blot Analysis of Protein Modifications

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Cell lysates were prepared in RIPA buffer and Western blots were performed as previously described (67 (link)). Primary antibodies were anti-PARK7 (Santa-Cruz, clone D4, 1 : 1000), anti-PGK1 (Proteintech 17811–1-AP, 1:4,000), anti-GAPDH (Proteintech 60004–1-Ig, 1:20,000), anti–β-actin (Sigma, clone AC-15, 1:5,000), antiphospho-p53 (Cell Signaling #9284; 1:1,000), anti–P-H2AX (Millipore; 1:1,000), anti-GLO1 (1:1,000) and antimethylglyoxal (recognizing the MG-H1 modification of arginines, Cell Biolabs STA-011, 1:5,000) diluted in Tris-buffered saline (150 mM NaCl, 20 mM Tris-Cl pH 7.4, 0.05% Tween-20) containing 2% bovine serum albumin (Sigma).

Heremans I.P., Caligiore F., Gerin I., Bury M., Lutz M., Graff J., Stroobant V., Vertommen D., Teleman A.A., Van Schaftingen E, & Bommer G.T. (2022). Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite. Proceedings of the National Academy of Sciences of the United States of America, 119(4), e2111338119.

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Protein Expression Analysis by Western Blot

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Western blot analysis was performed as previously described [19 (link)]. Primary antibodies against occludin (c-term), ZO-1, and claudin-5 and secondary antibodies, Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 555 donkey anti-rabbit, were purchased from Thermo Fisher Scientific. To detect the formation of AGE products with MG-modification, mouse anti-methylglyoxal monoclonal antibodies (STA-011; Cell Biolabs, San Diego, CA, USA) were used. Primary antibodies against p-mTOR, t-Akt, p-Akt, t-S6K1, p-S6K1, HIF-1α, COXIV, VDAC, and Parkin (Prk8) were purchased from Cell Signaling Technology (4695S, Danvers, MA, USA). Primary antibodies against t-mTOR and occludin (E-5) and the secondary antibody donkey anti-rabbit IgG-FITC were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibody against LC3B was purchased from Sigma Aldrich. Primary antibodies against Glo-1 and Glo-2 were purchased from Abcam. To counterstain the total protein in the gel, Coomassie Brilliant Blue staining was used as previously described [8 (link)].

Kim D., Kim K.A., Kim J.H., Kim E.H, & Bae O.N. (2020). Methylglyoxal-Induced Dysfunction in Brain Endothelial Cells via the Suppression of Akt/HIF-1α Pathway and Activation of Mitophagy Associated with Increased Reactive Oxygen Species. Antioxidants, 9(9), 820.

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Western Blot Analysis of Protein Modifications

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Cell extraction was performed in 1% SDS buffer containing protease and phosphatase inhibitors (Roche, Penzberg, Germany). Protein concentration is determined using bicinchoninic acid assay (Pierce, Carlsbad, CA). Twenty µg of protein were separated on 10 or 12.5% SDS-PAGE and transferred to PVDF membranes (Roche). Blocking was performed in 5% non-fat dried milk (Biorad, Hercules, CA) in TBS-Tween 0.1% for 1 h. Membranes were then incubated overnight at 4 °C with primary antibodies: Argpyrimidine (1/10,000, mAb6B), MG-H1 (1/1000, STA-011, Cell Biolabs), Nrf2 (1/1000, Ab62353, Abcam), GLO1 (1/1000, #02-14, BioMac, Leipzig, Germany), β-actin (1/5000, A5441, Sigma). Membranes were then incubated for 1 h in the presence of the appropriate secondary antibody coupled to horseradish peroxidase. Immunoreactive bands were detected using ECL Western Blotting substrate (Pierce). Bands quantification by densitometry and normalization to ß actin was performed using ImageJ software (NIH, Bethesda, MD).

Nokin M.J., Durieux F., Bellier J., Peulen O., Uchida K., Spiegel D.A., Cochrane J.R., Hutton C.A., Castronovo V, & Bellahcène A. (2017). Hormetic potential of methylglyoxal, a side-product of glycolysis, in switching tumours from growth to death. Scientific Reports, 7, 11722.

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Cardiomyocyte RyR2-AGE Colocalization

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Isolated cardiomyocytes were simultaneously labeled with anti-RyR2 antibody (1:50, ab196355 Abcam) and anti-AGE (MAGE, 1:50 STA-011 Cell Biolabs). The secondary antibodies were conjugated to proximity ligation assay (PLA) oligo probes (PLA probe rabbit PLUS [DUO 92002] and PLA probe mouse MINUS [DUO 92004], Sigma) and were hybridized for 1h, 37°C. z-planes were acquired with a spectral confocal microscope (FluoView-1000, Olympus) and the number of positive cross-reactivity spots was quantified in backgroundsubtracted images (Image J).

Ruiz-Meana M., Minguet M., Bou-Teen D., Miro-Casas E., Castans C., Castellano J., Bonzon-Kulichenko E., Igual A., Rodriguez-Lecoq R., Vázquez J, & Garcia-Dorado D. (2019). Ryanodine Receptor Glycation Favors Mitochondrial Damage in the Senescent Heart. Circulation, 139(7).

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