Fibronectin coated coverslips

Manufactured by Merck Group

Sourced in United States

Fibronectin-coated coverslips are laboratory equipment used in cell culture applications. They provide a surface coating that promotes cell attachment and growth. The core function of these coverslips is to support and facilitate the study of cells in controlled environments.

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Lab products found in correlation

Flouromount g, by Thermo Fisher Scientific (1 mentions) Axiovert 200, by Zeiss (1 mentions) 70 μm cell strainer, by Thermo Fisher Scientific (1 mentions) Collagenase type 2, by Merck Group (1 mentions) Geltrex, by Merck Group (1 mentions) C1 plus, by Nikon (1 mentions) Mouse anti ha antibody clone 16b12, by Fortrea (1 mentions) Nis elements software, by Nikon (1 mentions) Slowfade, by Thermo Fisher Scientific (1 mentions) Eclipse ti e inverted microscope system, by Nikon (1 mentions) Prolong mounting medium with dapi, by Thermo Fisher Scientific (1 mentions) Ab124824, by Abcam (1 mentions) Dynasore, by Selleck Chemicals (1 mentions) Anti cd164 sc 271179, by Santa Cruz Biotechnology (1 mentions) Nis elements ar 4, by Nikon (1 mentions) Hoechst 33256, by Merck Group (1 mentions) Cxcl12, by Abcam (1 mentions) A1r confocal microscope, by Nikon (1 mentions) Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Anti ssdna, by Abcam (1 mentions)

8 protocols using fibronectin coated coverslips

Visualizing Viral and Cellular Proteins

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Immunofluorescence assays of viral protein ICP4 were performed on cells grown on coverslips. Samples were fixed in 4% paraformaldehyde and incubated with the appropriate primary and secondary antibodies. DAPI (5 mg/ml) was added 10 min before the end of the procedure to visualize the nuclei. Cells were examined using a Zeiss Axiovert 200 fluorescence microscope. Images were captured by a Spot RT slider digital camera (Diagnostic) using MetaMorphTM imaging software, and processed using Adobe Photoshop CS4.
For confocal microscopy analysis of SAMHD1 subcellular localization, cells were grown on fibronectin-coated coverslips (20 µg/ml; Sigma) and fixed in 4% PFA. For SAMHD1 staining, blocking and staining solutions were composed of 0.2% Triton X-100, γ-globulin and TNB and appropriated primary and secondary antibodies. Samples were washed with TBS-Triton and mounted on coverslips with Flouromount-G (Thermo Fisher) containing DAPI (0.1 μM). Confocal images were obtained with an A1R + Nikon confocal microscopy attached to an inverted microscope (Eclipse Ti-E model, Nikon) with a Plan-Apocromatic 60X/1.4 oil immersion objective, using NIS Elements 4.40 acquisition software. Images were analysed with ImageJ.

Benayas B., Sastre I., López-Martín S., Oo A., Kim B., Bullido M.J., Aldudo J, & Yáñez-Mó M. (2020). Tetraspanin CD81 regulates HSV-1 infection. Medical Microbiology and Immunology, 209(4), 489-498.

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Isolation of Neonatal Cardiomyocytes

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All experiments were conducted in accordance with the United Kingdom Animals (Scientific Procedure) Act of 1986. NRVMs were established from wild-type and Snell's waltzer neonatal mouse pups. Surgically removed hearts were stored in ice-cold PBS and isolated ventricles were incubated in 0.5% trypsin-EDTA overnight with constant shaking. The following day, ventricles were warmed in NRVM medium (500 ml medium 199 plus 2 mM L-glutamine, 10 mM HEPES, 100 μM non-essential amino acids, 1.75 g glucose, 10 μg/ml vitamin B12 and penicillin-streptomycin) supplemented with 10% FBS and digested with type II collagenase (Sigma) (1 mg/ml in HBSS). Three collagenase fractions were added to HBSS on ice, centrifuged, resuspended and strained with a 70 μm cell strainer (Fisher). Single cells were resuspended in warm NRVM medium with 10% FBS and plated on a 10 cm dish to isolate fibroblasts. 90 min later, enriched cardiomyocytes were plated at a concentration of 10⁶ cells/well (for a 12-well dish) on fibronectin-coated coverslips (Sigma). On subsequent days, NRVMs were washed with NRVM medium supplemented with 2% FBS and imaged within 2 days.

Waxse B.J., Sengupta P., Hesketh G.G., Lippincott-Schwartz J, & Buss F. (2017). Myosin VI facilitates connexin 43 gap junction accretion. Journal of Cell Science, 130(5), 827-840.

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Blastoid-Derived Stem Cell Cultures

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Derivation experiments were performed with blastoids cultured for 96 h as described in the previous section. Blastoids were individually transferred on gelatin-coated 96-well plates with feeder layers of gamma-irradiated MEFs. Naive PSCs were derived in PXGL medium^{2 (link)}. TSCs were derived in human TSC medium^{20 (link)}. After 24 h of culture on feeders, blastoids attached and, within one week, colonies were formed. Derivation was considered successful after three passages after blastoid transfer. For immunofluorescence assays, naive PSCs were transferred onto Geltrex (0.5 µl cm⁻²)-coated coverslips, and TSCs were transferred onto fibronectin-coated coverslips (5 μg ml⁻¹, Sigma Aldrich, 08012).

Kagawa H., Javali A., Khoei H.H., Sommer T.M., Sestini G., Novatchkova M., Scholte op Reimer Y., Castel G., Bruneau A., Maenhoudt N., Lammers J., Loubersac S., Freour T., Vankelecom H., David L, & Rivron N. (2021). Human blastoids model blastocyst development and implantation. Nature, 601(7894), 600-605.

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Quantifying Bacterial Phagocytosis by Macrophages

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Transgenic BMDMs (1 × 10⁵) expressing HA-BAI1 were plated on fibronectin-coated coverslips (Sigma). The following day, the cells were incubated with E. coli DH5α–dsRed at an MOI of 10 for 30 min at 37°C. Cells were then fixed with 4% PFA and labeled with Alexa Fluor 647–conjugated WGA (5 mg/ml; Life Technologies) in Hanks’ balanced salt solution (HBSS) for an additional 10 min to label the plasma membrane. After washing, the cells were permeabilized for 30 min in PBS containing 3% BSA, 1% normal goat serum, FcR blocking antibody (clone 93; eBioscience), and 0.1% Triton X-100. Cells were labeled with mouse anti-HA antibody (clone 16B12; Covance) followed by Alexa Fluor 488–conjugated anti-mouse secondary antibody. ROIs for E. coli DH5α-treated cells were determined by dsRed signal. WGA signal was used to define ROIs in S. aureus conditions because the bacteria displayed substantially greater staining than did eukaryotic cell membranes. Images were captured with a Nikon C1 Plus confocal microscopewith z-stacks at 0.5μm. Analysis and processing were performed with NIS-Elements software (Nikon).

Billings E.A., Lee C.S., Owen K.A., D’Souza R.S., Ravichandran K.S, & Casanova J.E. (2016). The adhesion GPCR BAI1 mediates macrophage ROS production and microbicidal activity against Gram-negative bacteria. Science signaling, 9(413), ra14.

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Immunofluorescence Imaging of Adherent Cells

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IIF on fixed cellsSNU539/ S08-38710 and MAD10-252/616 cells were grown on fibronectin coated coverslips (Sigma, USA), and 36 hours later, cells were fixed with 4% paraformaldehyde in DPBS (PFA) for 15 min at room temperature, washed with DPBS and blocked with 5% NGS. PIM/IM antibodies diluted 1:200 were added to coverslips and incubated at room temperature for 2 hours. Following washes, a 1:500 GαRb Alexa 488 secondary antibody containing DAPI and red phalloidin (to stain for actin filaments) was added for 1 hour in the dark, washed and mounted on slides with Slowfade (Molecular Probes, USA), dried, and imaged.

Pires E.S., D'Souza R.S., Needham M.A., Herr A.K., Jazaeri A.A., Li H., Stoler M.H., Anderson-Knapp K.L., Thomas T., Mandal A., Gougeon A., Flickinger C.J., Bruns D.E., Pollok B.A, & Herr J.C. (2015). Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors. Oncotarget, 6(30), 30194-30211.

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Immunofluorescence Staining of Adherent Cells

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Cells were seeded in fibronectin-coated coverslips (Sigma Aldrich). When desired, cells were fixed in 2% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Blocking step was made in 3% Bovine Serum Albumin (BSA) for 1 h. Cells were incubated overnight at 4 °C with the primary antibody diluted in blocking buffer. Next day, secondary antibodies were incubated for 1 h at room temperature in the dark. Antibodies and dilutions are listed in Supplementary Table 7. Finally, cells were mounted in Prolong Mounting Medium with DAPI (Invitrogen) and images were taken in a Nikon Eclipse Ti-E inverted microscope system. For quantification, at least 200 cells per staining were evaluated using ImageJ software.

Boix O., Martinez M., Vidal S., Giménez-Alejandre M., Palenzuela L., Lorenzo-Sanz L., Quevedo L., Moscoso O., Ruiz-Orera J., Ximénez-Embún P., Ciriaco N., Nuciforo P., Stephan-Otto Attolini C., Albà M.M., Muñoz J., Tian T.V., Varela I., Vivancos A., Ramón y Cajal S., Muñoz P., Rivas C, & Abad M. (2022). pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation. Nature Communications, 13, 6840.

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CXCR4 and CD164 Colocalization Assay

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Cells seeded on fibronectin-coated coverslips (Sigma) were serum starved for 24 h and pretreated with 80 μM dynasore (S8047, Selleckchem, Houston, TX, USA), or DMSO as control, in 1% FBS medium for 30 min at 37°C. Cells were then stimulated with CXCL12 (100 ng/ml, Abcam) in 1% FBS medium for 5 min at 37°C. Cells were fixed in 4% paraformaldehyde, permeabilized in Triton X-100 0.15%-PBS, blocked in 4% BSA and incubated with the following primary antibodies: anti-CXCR4 (ab124824, dilution 1:100, Abcam); anti-CD164 (sc-271179, dilution 1:50, Santa Cruz Biotechnologies). Anti-rabbit rhodamine (#31686, dilution 1:100, Thermo Scientific) and anti-mouse FITC (#31569, dilution 1:100, Thermo Scientific) were employed as secondary antibodies. Nuclei were counterstained with Hoechst 33256 (Sigma). Confocal analysis was performed using Nikon A1R confocal microscope with a Plan Apo 60x/NA 1.4 DIC N2 objective (Nikon, Minato, Tokyo, JP). To determine colocalization of the proteins of interest, Z-stacks were acquired at 0.25 μm intervals using the following settings: 1,024 × 1,024 pixel, 2 scanner zoom, 0.5 μm scan speed. Images were analyzed using Nis Elements AR4.20.01 software (Nikon, Minato, Tokyo, JP). Colocalization was quantified by Mander's Colocalization Coefficient as we previously performed (31 (link)).

Mancarella C., Caldoni G., Ribolsi I., Parra A., Manara M.C., Mercurio A.M., Morrione A, & Scotlandi K. (2020). Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Modulates Aggressiveness of Ewing Sarcoma by Regulating the CD164-CXCR4 Axis. Frontiers in Oncology, 10, 994.

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Immunostaining of Single-Stranded DNA

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Cells were seeded on fibronectin-coated coverslips (Sigma-Aldrich). Fixation was performed in 80% methanol followed by 1 h blocking in PBS, 0.1% Tween, 1% BSA. ssDNA was visualized using anti-ssDNA (Abcam) at 1:50 dilution in PBS, 0.1% Tween, 1% BSA followed by incubation with anti-mouse Alexa 488-conjugated secondary antibody (Life Technologies). When indicated, S1 Nuclease digestion was performed before ssDNA staining. Nuclei were stained with DAPI and images were collected on Leica DM6000 or Zeiss Apotome microscopes.

Brégnard C., Guerra J., Déjardin S., Passalacqua F., Benkirane M, & Laguette N. (2016). Upregulated LINE-1 Activity in the Fanconi Anemia Cancer Susceptibility Syndrome Leads to Spontaneous Pro-inflammatory Cytokine Production. EBioMedicine, 8, 184-194.

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