Prostate cells pre-loaded with Calcein-AM were seeded at 2 × 104 cells/well in black tissue culture-treated 96-well plates (Corning) pre-coated with human plasma Fibronectin (FN) (Sigma-Aldrich) or basement membrane extract (Trevigen, MD, USA) at 10μg/mL and 0.5X respectively. Plates were centrifuged at 200 rcf for 2min, incubated for 1h and read using the FLUOstar-OPTIMA plate reader (490/520 nm) to obtain an initial intensity of total cell fluorescence. Non-adherent cells were removed and fluorescence intensity of adherent cells determined.
Human plasma fibronectin fn
Manufactured by Merck Group
Sourced in United States
Human plasma fibronectin (FN) is a high-molecular-weight glycoprotein found in blood plasma and the extracellular matrix. It plays a crucial role in cell adhesion, growth, migration, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Heparin, by Merck Group (5 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Basement membrane extract, by Bio-Techne (1 mentions)
Dmem ham s f12, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Cytoskeleton (1 mentions)
Alexa 488 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Radio immunoprecipitation assay ripa buffer, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Ecl plus system, by Cytiva (1 mentions)
Mounting medium for fluorescence with dapi, by Vector Laboratories (1 mentions)
8 protocols using human plasma fibronectin fn
1
Cell Adhesion Assay in Prostate Cells
2
PLLA-Borax Films for Cell Culture
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Cleaned glass cover slips were used as control substrates. PLLA (Cargill Dow) was dissolved at a concentration of 2% (w/v) in chloroform. Sodium Tetraborate Decahydrate Borax 10 Mol (hereafter borax) (Na2B4O7·10H2O, Borax España S.A) was dissolved in the culture medium in all the experiments. Two different borax concentrations were used, 0.59 mM and 1.47 mM, respectively, in the PLLA-B2% and PLLA-B5% samples. PLLA 2% (w/v) solution was used to prepare thin films by spin-coating on cleaned glass cover slips for 30 s at 3000 rpm. Samples were dried at 60 °C in vacuo for 4 h.
All the substrates were functionalized with human plasma fibronectin (FN, Sigma-Aldrich). After sterilizing with UV for 30 min, the substrates were coated with 20 µg mL−1 FN solution in Dulbecco’s Phosphate Saline Buffer (DPBS) for 1 h at room temperature.
All the substrates were functionalized with human plasma fibronectin (FN, Sigma-Aldrich). After sterilizing with UV for 30 min, the substrates were coated with 20 µg mL−1 FN solution in Dulbecco’s Phosphate Saline Buffer (DPBS) for 1 h at room temperature.
3
Isolation and Immortalization of Mouse Lung Microvascular Endothelial Cells
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Primary mouse lung microvascular endothelial cells (mLMEC) were isolated from WT C57/BL6 adult mice. mLMECs were twice subject to magnetic-activated cell sorting to positively select for endomucin+ ECs as previously described by Reynolds and Hodivala-Dilke (37 (link)). ECs were immortalized using polyoma-middle-T (PyMT) antigen by retroviral transfection as previously described by Robinson and colleagues (38 (link)). Immortalized mLMECs were cultured in media composed of a 1:1 mix of low-glucose Ham's F-12:DMEM (Thermo Fisher Scientific) medium supplemented with 10% FBS, 100 units/mL penicillin/streptomycin, 2 mmol/L glutamax, 50 μg/mL heparin (H3393, Sigma). ECs were grown at 37°C in a humidified incubator with 5% CO2 unless otherwise stated. For experimental analyses, plasticware was coated using 10 μg/mL human plasma fibronectin (FN; Millipore). Passaging of ECs did not exceed 18.
EC stimulation was achieved using 30 ng/mL VEGF-A164 (VEGF-A; mouse equivalent of VEGF-A165) after 3 hours incubation in serum-free medium (OptiMEM; Thermo Fisher Scientific). VEGF-A was made in-house as previously described by Krilleke and colleagues (39 (link)).
EC stimulation was achieved using 30 ng/mL VEGF-A164 (VEGF-A; mouse equivalent of VEGF-A165) after 3 hours incubation in serum-free medium (OptiMEM; Thermo Fisher Scientific). VEGF-A was made in-house as previously described by Krilleke and colleagues (39 (link)).
4
Isolation and Immortalization of Mouse Lung Microvascular Endothelial Cells
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Primary mouse lung microvascular endothelial cells (mLMECs) were isolated from age-matched (3–6 weeks) wild-type (WT) or floxed C57/BL6 mice. Cellular digests were expelled through a 19 gauge-needle and filtered through a 70 µm sterile strainer (Fisher Scientific). Cell pellets were seeded onto plasticware coated with a solution of 0.1% gelatin containing 10 µg/ml human plasma fibronectin (FN) (Millipore). and collagen type 1. mLMECs were twice positively selected for using endomucin primary antibody and magnetic activated cell sorting (MACS) as previously described by Reynolds & Hodivala-Dilke73 , prior to immortalisation using polyoma-middle-T-antigen (PyMT) as previously described by Robinson et al.74 (link).
ECs were cultured in a 1:1 mix of Ham’s F-12:Dulbecco’s Modified Eagle Medium (DMEM) (low glucose) medium supplemented with 10% foetal bovine serum (FBS), 100 units/mL penicillin/streptomycin (P/S) and 50 μg/mL heparin (Sigma) at 37 °C in a humidified incubator (+5% CO2) unless otherwise stated.
For experimental analyses, plasticware was coated using 10 µg/ml human plasma FN passaging of ECs did not exceed 20. VEGF-stimulation was achieved using 30 ng/ml VEGF-A164 (VEGF-A) (murine equivalent to VEGF-A165) post 3 h incubation in serum-free medium (OptiMEM®; Invitrogen). VEGF-A was made in-house, as previously described by Krilleke et al.75 (link).
ECs were cultured in a 1:1 mix of Ham’s F-12:Dulbecco’s Modified Eagle Medium (DMEM) (low glucose) medium supplemented with 10% foetal bovine serum (FBS), 100 units/mL penicillin/streptomycin (P/S) and 50 μg/mL heparin (Sigma) at 37 °C in a humidified incubator (+5% CO2) unless otherwise stated.
For experimental analyses, plasticware was coated using 10 µg/ml human plasma FN passaging of ECs did not exceed 20. VEGF-stimulation was achieved using 30 ng/ml VEGF-A164 (VEGF-A) (murine equivalent to VEGF-A165) post 3 h incubation in serum-free medium (OptiMEM®; Invitrogen). VEGF-A was made in-house, as previously described by Krilleke et al.75 (link).
5
Isolation and Immortalization of Mouse Lung Endothelial Cells
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Primary mouse lung microvascular endothelial cells (mLMECs) were isolated from adult mice bred on a mixed C57BL6/129 background. Primary ECs were twice positively selected for their expression of intracellular adhesion molecule-2 (ICAM-2) by magnetic activated cell sorting (MACS) as previously described by Reynolds and Hodivala-Dilke (Reynolds and Hodivala-Dilke, 2006 (link)). ECs were immortalized using polyoma-middle-T-antigen (PyMT) retroviral transfection as previously described by Robinson et al. (2009) (link). Immortalized mLMECs were cultured in IMMLEC media, a 1:1 mix of Ham’s F-12:DMEM medium (low glucose) supplemented with 10% FBS, 100 units/mL penicillin/streptomycin (P/S), 2 mM glutamax, 50 μg/mL heparin (Sigma). Immortalized mLMECs were cultured on 0.1% gelatin coated flasks at 37°C in a humidified incubator with 5% CO2. For experimental analyses, plates, dishes, flasks and coverslips were coated in 10 μg/ml human plasma fibronectin (FN) (Millipore) overnight at 4°C. Vascular endothelial growth factor-A (VEGF-A164: mouse equivalent of VEGF-A165) was made in-house as previously described by Krilleke et al. (2007) (link).
6
Investigating FAK Activation and EGFR Inhibition
7
Isolation and Immortalization of Primary Mouse Lung Microvascular Endothelial Cells
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Primary mouse lung microvascular endothelial cells (mLMECs) were isolated from adult mice bred on a pure C57/BL6 background. Primary ECs were twice positively selected for through their expression of endomucin by magnetic activated cell sorting (MACS) as previously described by Reynolds and Hodivala-Dilke. 27 ECs were immortalized using polyoma-middle-T-antigen (PyMT) retroviral transfection as previously described by Robinson et al 28 (link) Immortalized mLMECs were cultured in IMMLEC media, a 1:1 mix of Ham's F-12:DMEM medium (low glucose) supplemented with 10% FBS, 100 units/mL penicillin/streptomycin (P/S), 2 mM glutamax, 50 μg/mL heparin (Sigma).
Immortalized mLMECs were cultured on 0.1% gelatin coated flasks at 37°C in a humidified incubator with 5% CO 2 . For experimental analyses, plates, dishes and flasks were coated in 10 µg/mL human plasma fibronectin (FN) (Millipore) overnight at 4°C. Vascular endothelial growth factor-A (VEGF-A 164 : mouse equivalent of VEGF-A 165 ) was made in-house as previously described by Krilleke et al. 29 (link)
Immortalized mLMECs were cultured on 0.1% gelatin coated flasks at 37°C in a humidified incubator with 5% CO 2 . For experimental analyses, plates, dishes and flasks were coated in 10 µg/mL human plasma fibronectin (FN) (Millipore) overnight at 4°C. Vascular endothelial growth factor-A (VEGF-A 164 : mouse equivalent of VEGF-A 165 ) was made in-house as previously described by Krilleke et al. 29 (link)
8
Isolation and Immortalization of Mouse Lung Microvascular Endothelial Cells
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Primary mouse lung microvascular endothelial cells (mLMECs) were isolated from adult mice bred on a pure C57/BL6 background. Primary ECs were twice positively selected for through their expression preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted January 17, 2021. ; https://doi.org/10.1101/2021.01.15.426835 doi: bioRxiv preprint 13 of endomucin by magnetic activated cell sorting (MACS) as previously described by Reynolds & Hodivala-Dilke (Reynolds and Hodivala-Dilke, 2006) . ECs were immortalised using polyoma-middle-Tantigen (PyMT) retroviral transfection as previously described by Robinson et al (Robinson et al., 2009) (link). Immortalised mLMECs were cultured in IMMLEC media, a 1:1 mix of Ham's F-12:DMEM medium (low glucose) supplemented with 10% FBS, 100 units/mL penicillin/streptomycin (P/S), 2 mM glutamax, 50 μg/mL heparin (Sigma).
Immortalised mLMECs were cultured on 0.1% gelatin coated flasks at 37°C in a humidified incubator with 5% CO2. For experimental analyses, plates, dishes and flasks were coated in 10 µg/ml human plasma fibronectin (FN) (Millipore) overnight at 4°C. Vascular endothelial growth factor-A (VEGF-A164: mouse equivalent of VEGF-A165) was made in-house as previously described by Krilleke et al. (Krilleke et al., 2007) (link).
The copyright holder for this this version posted January 17, 2021. ; https://doi.org/10.1101/2021.01.15.426835 doi: bioRxiv preprint 13 of endomucin by magnetic activated cell sorting (MACS) as previously described by Reynolds & Hodivala-Dilke (Reynolds and Hodivala-Dilke, 2006) . ECs were immortalised using polyoma-middle-Tantigen (PyMT) retroviral transfection as previously described by Robinson et al (Robinson et al., 2009) (link). Immortalised mLMECs were cultured in IMMLEC media, a 1:1 mix of Ham's F-12:DMEM medium (low glucose) supplemented with 10% FBS, 100 units/mL penicillin/streptomycin (P/S), 2 mM glutamax, 50 μg/mL heparin (Sigma).
Immortalised mLMECs were cultured on 0.1% gelatin coated flasks at 37°C in a humidified incubator with 5% CO2. For experimental analyses, plates, dishes and flasks were coated in 10 µg/ml human plasma fibronectin (FN) (Millipore) overnight at 4°C. Vascular endothelial growth factor-A (VEGF-A164: mouse equivalent of VEGF-A165) was made in-house as previously described by Krilleke et al. (Krilleke et al., 2007) (link).
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