A11013

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A11013 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for performing specific laboratory tasks, but a detailed and unbiased description of its core function cannot be provided without the risk of interpreting or extrapolating beyond the available information. As a Marketing Specialist, I aim to present facts objectively without making assumptions about the intended use of this product.

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20 protocols using a11013

Indirect Immunofluorescence of Angiogenic Markers

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For indirect immunofluorescence the cells were seeded on cover slips at density of 20x10³ cells/well, overnight. When necessary, the cells were incubated during 24-48h with 2mg/ml Beva in DMEM 0.5% FBS. Then, cells were fixed and permeabilized in methanol during 20min. After blocking with 5% bovine serum albumin (BSA) for 30min, cells were incubated overnight at room temperature with the primary antibodies. Then, 1h of incubation with the secondary antibody anti-Human IgG-Alexa Fluor 488 (1:500 dilution, A11013, Invitrogen) in 5% BSA was performed for Beva; anti-rabbit-Alexa Fluor 568 (1:500 dilution, A11011, Invitrogen) in 5% BSA was performed for VEGF; anti-rabbit-Alexa Fluor 488 (1:500 dilution, A11008, Invitrogen) in 5% BSA was performed for MCT4, MCT1 and GLUT-1 and anti-mouse-Alexa Fluor 594 antibody (1:250 dilution, A11032, Invitrogen) for CD147, HKII, LDHA and CAXI. Additionally, to see the internalization of Beva, the cells exposed to Beva during 24h, we directly incubated with an anti-Human IgG-Alexa Fluor 488 (1:500 dilution, A11013, Invitrogen) after fixation and blocking.
Finally, after washing in PBS, cells were mounted in Vectashield Mounting Media with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and images were obtained with a fluorescence microscope (Olympus IX81), using the Cell P software.

Miranda-Gonçalves V., Cardoso-Carneiro D., Valbom I., Cury F.P., Silva V.A., Granja S., Reis R.M., Baltazar F, & Martinho O. (2017). Metabolic alterations underlying Bevacizumab therapy in glioblastoma cells. Oncotarget, 8(61), 103657-103670.

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Antibody Measurement of PfEMP1-VarO Proteins

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Antibodies (IgG) to PfEMP1-VarO protein displayed on iRBC surface were monitored by indirect immuno-fluorescence using flow cytometry or fluorescence microscopy. After rosette enrichment, an aliquot of 89F5 VarO rosetting culture (>85% rosetting rate) was resuspended at 2.5% hematocrit in DPBS/FCS medium (DPBS, 2% foetal calf serum). Parasite suspension (20–40 μL) was incubated 30 min at 37°C with plasma or serum diluted 1/20. After 2 washes with DPBS/FCS, the cell pellet was resuspended in 100 μL DPBS/FCS medium containing goat anti human IgG (H+L) Alexa Fluor 488 conjugate (Molecular probes, A-11013, diluted 1/1,000) and Hoechst 33342 (Molecular probes, H-3570, diluted 1/1,000). After an incubation of 30 min at 37°C, parasite pellets were washed twice with DPBS/FCS medium and resuspended in 200 μL formaldehyde 0.37% (Sigma F-8775). Immuno-fluorescence staining was analysed as described [33 (link)].

Guillotte M., Juillerat A., Igonet S., Hessel A., Petres S., Crublet E., Le Scanf C., Lewit-Bentley A., Bentley G.A., Vigan-Womas I, & Mercereau-Puijalon O. (2015). Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes. PLoS ONE, 10(7), e0134292.

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Quantifying Plasma Cell Enumeration

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Cultured cells (5× 10⁴ - 5× 10⁵) were cytocentrifuged onto poly-L-lysine-coated slides (for 5 minutes at ~120 g; Cyto-Tek centrifuge model 4332; Sakura Finetek, Japan). Slides were air-dried for 1 hour at 22°C and then fixed in 4% paraformaldehyde at 4°C for 10 minutes. Subsequently, they were blocked with 2% bovine serum albumin in PBS and incubated with Hoechst 33342 solution (2 μg/mL, Cat. B2261; Sigma-Aldrich) to stain DNA. We stained for plasma cells with mouse anti-human CD138 mAb (1:250, Clone MI15, Dako), donkey anti-mouse IgG Alexa 594 (1:300, A21203; Molecular Probes-Invitrogen) and goat anti-human IgG Alexa 488 (1:500, A11013; Molecular Probes-Invitrogen). Slides were mounted in 80% glycerol-TBS and stored at 4°C in the dark. All washing and incubation steps were performed with TBSTriton X-100 (0.03%). We counted the plasma cells from each well, in a blinded fashion, on a fluorescence microscope (Olympus BX51), identifying them by their distinctive size, shape (extensive cytoplasm and eccentric nuclei), and positive staining for internal IgG and/or surface CD138. Absolute numbers of plasma cells per well are given in Table 2 and shown in the Figures as the % of those in untreated cultures. Total numbers of recovered cells were measured by automated counting of trypan blue-excluding cells (TC20 Automated Cell Counter, BioRad).

Gomez A.M., Willcox N., Vrolix K., Hummel J., Nogales-Gadea G., Saxena A., Duimel H., Verheyen F., Molenaar P.C., Buurman W.A., De Baets M.H., Martinez-Martinez P, & Losen M. (2014). Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients. Journal of immunology (Baltimore, Md. : 1950), 193(3), 1055-1063.

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Tanycyte Leptin Trafficking Assay

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Tanycytes were seeded on poly-L-lysine-coated glass coverslips (10μg/ml) and incubated in TDM (DMEM/F-12, #11039, ThermoFisher), 1% L-glutamine (#25030-024, ThermoFisher), 2% penicillinstreptomycin (#P4458, Sigma), insulin (1l1000, #I5500, Sigma), putrescine (1l500, #P5780, Sigma)) for 24h before the experiment. Tanycytes were incubated for the indicated time at 37°C with either bioactive fluorescent leptin (Fluorescent-leptin, 125nM, Cisbio Bioassays) or a fluorescent leptin antagonist (LAN, 125nM, Cisbio Bioassays), both diluted in TDM. Cells were then fixed for 10min at 4°C with 4% paraformaldehyde and washed thrice with PBS 1X. For cointernalization, tanycytes were incubated with Fluorescent-leptin and XPA (30nM in TDM, Xoma Laboratories) for 5min, washed and fixed. Cells were then washed and permeabilized with 0.1% TritonX-100 (v/v in PBS) for 5min at room temperature. All antibodies were diluted in PBS-3% BSA and incubated for 45min at room temperature: anti-EEA1 (1l200; Santa Cruz Biotechnology Cat#sc-641 5, RRID:AB_2096822) followed by AlexaFluor-488-conjugated anti-goat (1/1000; Molecular Probes Cat#A-11055, RRID:AB_2534102) or AlexaFluor-488-conjugated anti-human antibodies (1/1000; Molecular Probes Cat#A-11013, RRID:AB_141360) to label XPA.

Duquenne M., Folgueira C., Bourouh C., Millet M., Silva A., Clasadonte J., Imbernon M., Fernandois D., Martinez-Corral I., Kusumakshi S., Caron E., Rasika S., Deliglia E., Jouy N., Oishi A., Mazzone M., Trinquet E., Tavernier J., Kim Y.B., Ory S., Jockers R., Schwaninger M., Boehm U., Nogueiras R., Annicotte J.S., Gasman S., Dam J, & Prévot V. (2021). Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function. Nature metabolism, 3(8), 1071-1090.

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Tanycyte Leptin Trafficking Assay

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Quantitative Analysis of Vaccinia Virus Antigens

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Briefly, 2 × 10⁶ of HEK293T cells were transfected with 5 μg ALAB mRNA or 5 μg 4Sin mRNA using TransIT™-mRNA Transfection Kit (Mirus Biotech, Madison, WI, USA, MIR-2250) or with transfection Kit alone. After 24 h culturing, cells were detached from the six-well plate surface with PBS containing 3% FBS, followed by staining with aqua fluorescent reactive dye (Invitrogen, L34966A, 1:1000) to differentiate alive versa dead. Cells were fixed with IC Fixation Buffer (Invitrogen, 00-8222-49), permeabilized (Invitrogen, 008333-56) and blocked with CD16/CD32 monoclonal antibody (Invitrogen, 14-0161-85, 1:1000). Finally, the cells were stained with monoclonal antibodies (Henxy Biotech, Shanghai, China, RAB-2412, RAB-2422, RAB-2471, RAB-2442) that specifically recognizes A27, L1, A33, or B5 (1:200 dilution), respectively, followed by adding anti-mouse AF488 (Invitrogen, A55058, 1:500) or anti-human AF488 (Invitrogen, A-11013 1:500) as secondary antibodies. Cells were acquired on a FACS Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) and data were analyzed using FlowJo 10.8.1.

Su C., Li S., Wen Y., Geng X., Yin Q., Wang Y., Xiong Y, & Liu Z. (2024). A Quadrivalent mRNA Immunization Elicits Potent Immune Responses against Multiple Orthopoxviral Antigens and Neutralization of Monkeypox Virus in Rodent Models. Vaccines, 12(4), 385.

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Siglec-15 Expression Analysis

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Hek293-hsiglec-15 or U87 cells were harvested and incubated with anti-Siglec-15 mAbs (from 0 to 10,000 ng/mL or 0 to 45,000 ng/mL) for 1 hour at 4°C. After incubation, the samples were washed with PBS 3 times and stained with Alexa488-conjugated anti-human (Invitrogen #A-11013) or mouse (Invitrogen #A32723) secondary antibodies in the dark for 50 minutes at 4°C. The mean fluorescence intensity of cells was measured by FACScan (Thermo). The data were analyzed using FlowJo software.

Ding H., Yao B., Ci L., Feng J., Ouyang P., Chen G., Hui X, & Zhou D. (2023). Enhancing the Anti-tumor Potency of a Novel Siglec-15 Antibody by Engineering its Fc-mediated Effector Functions. Journal of Immunotherapy (Hagerstown, Md. : 1997), 46(5), 161-169.

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Immunofluorescence Microscopy of Ovarian Cancer Cells

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The immunofluorescent microscopy was performed using methods previously described.⁷⁷ (link) Briefly, 1 × 10⁵ ovarian cancer cells (OVCAR3, OVCAR4 and PEO1) and negative control cells (OSEC) were seeded on coverslips and incubated overnight. The cells on coverslips were fixed in 4% PFA and incubated overnight with IgG antibodies extracted from ovarian tumour tissues diluted 1:100 in PBS and 1% FCS. The coverslips were then incubated with 1:200 anti-human IgG (Invitrogen, A-11013) and mounted with DAPI (Invitrogen, P36935). The images were taken using confocal microscope (Leica SP5). The confocal images were analysed using Fiji.

Lu H., Lou H., Wengert G., Paudel R., Patel N., Desai S., Crum B., Linton-Reid K., Chen M., Li D., Ip J., Mauri F., Pinato D.J., Rockall A., Copley S.J., Ghaem-Maghami S, & Aboagye E.O. (2023). Tumor and local lymphoid tissue interaction determines prognosis in high-grade serous ovarian cancer. Cell Reports Medicine, 4(7), 101092.

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Immunofluorescence Analysis of Viral Coinfection and Antibody Treatment

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Vero E6 cells (2 × 10⁴) were cultured in 8-well chamber slides. The hCoV-OC43 and adapted-H1N1 co-infection and 3D8 scFv treatment were performed as described above. The slides were fixed with cold-methanol and permeabilized with an Intracellular Staining Perm Wash Buffer (Biolegend, USA) for 15 min each. Following blocking with PBS with 0.1% tween 20 containing 1% BSA and glycine for 1 h, the cells were incubated with polyclonal rabbit anti-HA antibody (PA5-349291, Invitrogen, USA), monoclonal anti-coronavirus antibody (OC43 strain, clone 541-8F, MAB9012, Sigma-Aldrich, USA), and anti-3D8 antibody (humanized antibody, clone 1D7, Bioneer, Republic of Korea) at 1:1000 dilution for 24 h at 4 °C. After that, goat anti-human IgG Alexa fluor 488 (A-11013, Invitrogen, USA), donkey anti-rabbit IgG Alexa fluor 555 (ab150074, Abcam, UK), and goat anti-mouse IgG Alexa fluor 647 (ab1500115, Abcam, UK) were incubated for 1 h at 25 °C. The nucleus was stained with VECTASHIELD Antifade mounting medium containing DAPI (LSbio, USA) and visualized using a Zeiss LSM 900 confocal microscope (Zeiss, German). The viral protein signals were converted to relative intensity percentages using CellProfiler 4.2.1, and the viral protein intensity was normalized to DAPI intensity.

Luong Q.X., Hoang P.T., Lee Y., Ayun R.Q., Na K., Park S., Lin C., Ho P.T., Lee T.K, & Lee S. (2024). An RNA-hydrolyzing recombinant minibody prevents both influenza A virus and coronavirus in co-infection models. Scientific Reports, 14, 8472.

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Immunolabeling of Nucleolar Proteins in Cells

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The cells were fixed with 4% (weight/volume) paraformaldehyde in PBS for 15 min, washed with PBS, solubilized in 0.5% (volume/volume) Triton X-100 (Sigma) in PBS for 10 min and washed again before being incubated in primary antibodies that included: fibrillarin (Sigma, AnA-N), rabbit POLRIE/PAF53 antibody at 1:100 (Proteintech, 16145-1-AP), anti-UBF (provided by Edward Chen at the University of Florida) at 1:600. Incubations were for 1 h before cells were washed, and then finally incubated in anti-human, anti-mouse, or anti-rabbit Alexa-fluor secondary antibodies at 1:200 (Invitrogen, A-11013, A-11014, A-11001, A-11005) for 1 h. The cells were then mounted using Vectashield antifade mounting media with DAPI (Vector Laboratories Inc, H-1200). Immunolabeled cells were imaged using a Nikon Eclipse Ti fluorescence microscope with 60X objective and 1.4NA and 4 channels of fluorescence.

McNamar R., Freeman E., Baylor K.N., Fakhouri A.M., Huang S., Knutson B.A, & Rothblum L.I. (2023). PAF49: An RNA Polymerase I subunit essential for rDNA transcription and stabilization of PAF53. The Journal of Biological Chemistry, 299(8), 104951.

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