Attachment factor

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Attachment Factor is a laboratory equipment used to facilitate the attachment of cells or other biological samples to various surfaces. It provides a stable and uniform surface for cell growth and interaction studies. The Attachment Factor functions by enhancing the adhesion of cells to the designated substrate, enabling researchers to conduct experiments and observations in a controlled environment.

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Attachment factor protein (7 citations) Attachment Factor Protein 1X (2 citations)

24 protocols using attachment factor

Culturing Human Endothelial Cells

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Human pulmonary artery endothelial cells (HPAECs) were purchased from PromoCell (Switzerland). HPAECs were cultured in T25 flasks, 6‐well plates or on glass coverslips coated with attachment factor (Gibco), and allowed to grow and reach confluence in endothelial cell growth medium 2 (PromoCell, Switzerland) containing endothelial cell growth medium supplement 2 (PromoCell, Switzerland) and Gentamicin/amphotericin (Gibco).
Human umbilical vein endothelial cells (HUVECs) were purchased from Gibco. HUVECs were cultured in T25 flasks, 6‐well plates or on glass coverslips coated with attachment factor (Gibco), and allowed to grow and reach confluence in medium 200 containing low serum growth supplement and Gentamicin/amphotericin (Gibco).
Cells from passage 3 to 6 were used for all experiments.

Beddek K., Raffin F., Borgel D., Saller F., Riccobono D., Bobe R, & Boittin F. (2021). TRPV4 channel activation induces the transition of venous and arterial endothelial cells toward a pro‐inflammatory phenotype. Physiological Reports, 9(3), e14613.

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Cell Culture Protocols for Drosophila, Mouse ESCs, and Human Fibroblasts

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Drosophila S2 cells (Thermo Fisher R69007) were cultured at 25 °C in serum free medium (Express Five SFM, Gibco), supplemented with 20 mM l-Glutamin (GlutaMAX, Gibco). Cells were passaged with 10% of conditioned medium each passage. Cells were maintained at a density of 1–12million ml⁻¹.
Mouse embryonic stem cells were cultured in a humidified incubator at 37 °C and 5% CO₂. Cells were grown on Attachment Factor (Thermo Fisher) in 2i medium (KnockOut^TM Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 15% KnockOut^TM Serum Replacement (Gibco), 0.1 mM MEAA (Gibco, non-essential aminoacid), 1 nM Na-Pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 4 mM l-Glutamin (GlutaMAX, Gibco), 5 μg ml⁻¹ Insulin (Sigma), 50 U ml⁻¹ Pen-Strep, 200 U ml⁻¹ LIF (ESGRO), 1 μM PD0325901 (StemGent), 3 μM CHIR99021 (StemGent).
Human fibroblast cell lines were established from skin biopsies of confirmed Koolen de-Vries syndrome patients and healthy controls. Cells were maintained in in a humidified incubator at 37 °C and 5% CO2 in DMEM (GlutaMAX supplement, Life Technologies), 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 10% foetal calf serum (FCS). All fibroblast lines tested negative for Hepatitis B, Hepatitis C, HIV and human Herpes virus 4 and 8. Fibroblasts were passaged at ~90% confluency.

Gaub A., Sheikh B.N., Basilicata M.F., Vincent M., Nizon M., Colson C., Bird M.J., Bradner J.E., Thevenon J., Boutros M, & Akhtar A. (2020). Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation. Nature Communications, 11, 2243.

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Thrombin-Induced Endothelial Cell Permeability

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ECs were grown on a coverslip coated with attachment factor (Thermo Fisher Scientific, Waltham, MA). Once cells became 85–90% confluent, they were serum starved for 4 h and then treated with different inhibitors for 30 min before 2 units of thrombin were added. At the end, cells were fixed with 2% paraformaldehyde for 20 min and permeabilized with 0.1% triton-X-100 (Sigma-Aldrich, St. Louis, MO) for 5 min. Cells were blocked with 2% BSA in PBS and incubated with primary antibodies (anti–ZO-1, anti-VE-cadherin, anti-JAM-A, vinculin and anti-P-MLC antibody) in a blocking solution at 4 °C overnight. Th next day, after three washes with PBS, cells were incubated with corresponding secondary antibody for 1 h at room temperature. FITC-conjugated phalloidin was used to stain F-actin for 1 h at room temperature. After 8 washes with PBS, coverslips were mounted with Vecta-Shield (Hardset) mounting medium with DAPI (Vector Labs, Burlingame, CA). All images were captured with a Zeiss Microscope (Thornwood, NY) or EVOs microscope (Thermo Scientific, Philadelphia, PA). For quantitative analysis of thrombin-induced gap formation, the area covered by the cells was measured with ImageJ software at 5 randomly selected fields from three independent experiments.

Giri H., Srivastava A.K, & Naik U.P. (2022). Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability. Vascular pharmacology, 145, 107088.

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Endothelial Cell-Monocyte Interaction Assay

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Human umbilical vein vascular endothelial cells and monocyte/macrophage type 1 (THP-1) cells were acquired from the Chinese Academy of Sciences Cell Bank. High-glucose Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were sourced from Wisent Inc. Gibco fetal bovine serum (FBS), trypsin, and attachment factor were sourced from Thermo Fisher Scientific Inc. Cell senescence β-galactosidase staining kits were purchased from Beyotime Biotechnology. Evans blue (EB) and collagen were sourced from Sigma-Aldrich Corp. In vitro angiogenesis assay kits, Millicell hanging cell culture inserts, and 24- and 96-well plates were purchased from Millipore Sigma Co Ltd., and HTO (5 mCi) was obtained from PerkinElmer Life and Analytical Sciences Inc. E-Plate 16× was sourced from ACEA Bioscience Inc. Dimethyl sulfoxide was obtained from Beijing Solarbio Science and Technology Co, Ltd., and anti-Interleukin (IL)8, goat anti-rabbit, and goat anti-mouse antibodies were all from Abcam Plc.

Yan H.B., Liu Y.T., Li Z.Y., Wu Z.J., Zhang M., Xue P.J., Liu Y.L., Wang K.Z., He Y.M., Tu Y., Cui F.M, & Chen Q. (2020). Tritiated Water Induces Toxicity in Human Umbilical Vein Vascular Endothelial Cells via IL8. Dose-Response, 18(3), 1559325820938541.

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Assessing Paracrine Effects of BeWo Cells

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24 hr post-transfection, media on transfected BeWo cells was replaced with EGM-2 (Lonza, Basel, Switzerland). 48 hr post-transfection, BeWo cells were harvested for RNA analysis while the media was removed and placed on HPMVECs at 70% confluence grown in 6-well plates coated with attachment factor (Thermo Fisher Scientific). In a subset of BeWo cells, media and cells were collected for PGF ELISA analysis (R&D Systems, DPG00). ELISA data were normalized to total protein. HPMVECs were cultured in BeWo cell conditioned medium for 48 hr prior to harvesting for RNA analysis.

Lamm K.Y., Johnson M.L., Baker Phillips J., Muntifering M.B., James J.M., Jones H.N., Redline R.W., Rokas A, & Muglia L.J. (2018). Inverted formin 2 regulates intracellular trafficking, placentation, and pregnancy outcome. eLife, 7, e31150.

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Transwell Assay for Endothelial Cell Migration

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The migratory response of ECs was measured by transwell assay. In brief, transwell filter polycarbonate inserts with 0.4 μm pore size and 12 mm diameter (Corning, New York) were coated with attachment factor (Thermo Fisher Scientific, Waltham, MA). HUVECs (3 × 10⁴ cells/well) were seeded on the upper compartment and grown to confluence for 72h. Cells were treated in xCELLigence system, as mentioned, except fluorescein isothiocyanate (FITC)-dextran (40kDa, 1mg/ml;) was added before 5 min of thrombin treatment. From the lower compartment, 10 μl samples were taken after 20, 40, and 60min and diluted to 90 μl of basal medium. Finally, all diluted samples were read in a clear bottom fluorescence 96-well black plate at (excitation: 485nm; emission: 535nm) in a plate reader (Perkin Elmer). Control was set at 1 and the final graph plotted as fold change relative to the control.

Giri H., Srivastava A.K, & Naik U.P. (2022). Apoptosis signal-regulating kinase-1 regulates thrombin-induced endothelial permeability. Vascular pharmacology, 145, 107088.

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Adenoviral Infection of HUVECs for Immunofluorescence

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HUVECs were plated onto glass coverslips precoated with attachment factor (Life Technologies). The following day cells were infected with the adenoviral constructs and after an overnight incubation, the medium was replaced with fresh endothelial basal medium containing 10% fetal bovine serum. Forty-eight hours after infection, cells were rinsed 3× with PBS and fixed in 4% paraformaldehyde for 10 minutes followed by permeabilization with 0.1% Triton X-100 in PBS for 10 minutes. Cells were blocked in 1% BSA for 30 minutes at room temperature and incubated overnight at 4°C with C-19 antibody in PBS containing 1% BSA, or normal goat IgG for the negative control. After washing with PBS, cells were then incubated for 1 hour in the dark with Alexa Fluor 488-conjugated donkey anti-goat IgG (Life Technologies). Cells were then rinsed 3× with PBS then mounted using ProLong Gold antifade reagent with DAPI (Life Technologies). Images were acquired using a Leica TCS SP2 confocal microscope using a Planapo 63×/1.25 oil immersion objective and images were acquired in the horizontal (x-y) and in the vertical (x-z) planes by the LAS-AF software. Offline analysis was performed using ImageJ.

Mehta V., Fields L., Evans I.M., Yamaji M., Pellet-Many C., Jones T., Mahmoud M, & Zachary I. (2018). VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy-Terminal NRP1 Fragments That Regulate Angiogenic Signaling. Arteriosclerosis, Thrombosis, and Vascular Biology, 38(8), 1845-1858.

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Culturing Microvascular Endothelial Cells

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The CMECs are seeded on TC plates that are pre-coated for an hour with attachment factor (basically gelatin), from Life Technologies. The confluent plates are trypsinized with 0.25% Trypsin-EDTA solution (Whitehead Scientific, Cat no. BE 02-007E) and suspended cells are removed and re-plated in a 1:2 ratio. Suspended cells are centrifuged at 1000 rpm for 3 min to obtain a pellet. The pellet is re-suspended in a specialized growth medium. Microvascular Endothelial Cell Growth Medium-2 (Whitehead Scientific-Lonza^®, Cat no. CC-3156) and supplemented with the Bullet Kit (Whitehead Scientific-Lonza^®, Cat no. CC-4147), when ordered together as one package, the Cat no. is CC-3202.

Mentor S, & Fisher D. (2021). High-Resolution Insights Into the in vitro Developing Blood-Brain Barrier: Novel Morphological Features of Endothelial Nanotube Function. Frontiers in Neuroanatomy, 15, 661065.

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Evaluating HUVEC Invasion with Inhibitors

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Human umbilical vein endothelial cells (HUVEC, EMD Millipore, Billerica, MA) were cultured in EndoGRO-LS media (EMD Millipore) and maintained at 5% CO₂, 37°C, in 75 cm² vented cap flasks (Thermo-Fisher, Pittsburgh, PA). For invasion assays, HUVEC were plated at 1×10⁵ cells/ml in 35 mm culture dishes (Thermo-Fisher) or at 1×10⁴ cells/ml in 96-well plates (Thermo-Fisher) coated with Attachment Factor (Life Technologies, Carlsbad, CA). The next day, HUVEC were pretreated in medium containing the vehicle control, ML 141, or RSM structural analog. ML 141 and RSM structural analogs were suspended in dimethyl sulfoxide (DMSO, Thermo Fisher Scientific) or in polyethylene glycol (PEG, Sigma-Aldrich, St. Louis, MO) at a concentration of 5 mmol/L. The 5 mmol/L solution was diluted to 1 mmol/L in the solvent and then diluted to the final concentration for each experiment in EndoGRO-LS media. For vehicle control treatment, the same volume of DMSO or of PEG as that of ML 141 or of the RSM structural analog was added to medium. In most experiments, the invasion assay was performed 18-20 h later. Because in vitro data indicate that ML 141 is a reversible inhibitor [22 ], bacteria were added directly to media containing vehicle control, ML 141, or the RSM structural analog.

Cordero D., Fullenkamp C.R., Pelly R.R., Reed K.M., Caffo L.M., Zahrt A.N., Newman M., Komanapalli S., Niemeier E.M., Bishop D.L., Bruns H.A., Haynes M.K., Sklar L.A., Sammelson R.E, & McDowell S.A. (2014). Small Molecule Inhibitors Limit Endothelial Cell Invasion by Staphylococcus aureus. Current pharmaceutical biotechnology, 15(8), 727-737.

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Inactivation of NIH-3T3 Cells with Mitomycin C

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NIH-3T3 cells (DSMZ) were cultured in DMEM HG (Gibco) supplemented with FBS (10%; Biowest), penicillin/streptomycin (1%) and gentamycin (0.2%). Cells were seeded onto attachment factor (Life Technologies) coated plates at density 1.8×10⁵/well. The following day, cells were inactivated with mitomycin C (2 h; 15 μl/ml; Cayman Chemical).

Janik K., Popeda M., Peciak J., Rosiak K., Smolarz M., Treda C., Rieske P., Stoczynska-Fidelus E, & Ksiazkiewicz M. (2016). Efficient and simple approach to in vitro culture of primary epithelial cancer cells. Bioscience Reports, 36(6), e00423.

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