Comprehensive Analysis of Cell Proliferation, Motility, and Angiogenesis

Cell proliferation was determined with a WST-8 kit (Dojindo, Japan). Motility was determined by wound healing assays (16 (link)). In vitro angiogenesis and lymphangiogenesis were determined by tube formation assays as previously described (17 (link),18 (link)). QPCR, and western blotting were carried out essentially as described previously (10 (link)). Lipids were extracted and sphingolipids quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) (10 (link),19 (link)). Cells were fixed for 5 min in 4% paraformaldehyde in phosphate-buffered saline and blocked by horse serum, and immunocytochemistry was performed using the following primary antibodies: anti-ABCB1 (C219, Abcam, UK), anti-ABCC1 (MRPr1, Monosan, The Netherlands), and anti-SphK1 phospho-Ser225 (ECM Biosciences). The specificities of anti-ABCC1 and anti-SphK1 antibodies and anti-phospho-SphK1 specific antibody, phospho-Ser225, were previously confirmed using siRNA knockdown (10 (link),20 ). After incubation with biotinylated secondary antibodies, antigens were visualized with 3,30-diaminobenzidine (Dako, Denmark) and cells counterstained with hematoxylin.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Yamada A., Nagahashi M., Aoyagi T., Huang W.C., Lima S., Hait N.C., Maiti A., Kida K., Terracina K.P., Miyazaki H., Ishikawa T., Endo I., Waters M.R., Qi Q., Yan L., Milstien S., Spiegel S, & Takabe K. (2018). ABCC1-exported Sphingosine-1-phosphate, Produced by Sphingosine Kinase 1 Shortens Survival of Mice and Patients with Breast Cancer. Molecular cancer research : MCR, 16(6), 1059-1070.

Publication 2018

WST-8 kit 3,30-diaminobenzidine

Abcb1 Abcc1 Angiogenesis Anti antibodies Anti antibody Antibodies Antigens Assays Cell proliferation Cells Electrospray ionization mass spectrometry Hematoxylin Horse Immunocytochemistry Lipids Liquid chromatography Lymphangiogenesis Monosan Motility Paraformaldehyde Phosphate Saline Serum Sirna Sphingolipids Wst 8

Corresponding Organization : University at Buffalo, State University of New York

Other organizations : Yokohama City University Medical Center, Chang Gung Memorial Hospital

Top 5 similar protocols

Variable analysis

independent variables

Cell proliferation was determined with a WST-8 kit (Dojindo, Japan)
Motility was determined by wound healing assays (16 (link))
In vitro angiogenesis and lymphangiogenesis were determined by tube formation assays as previously described (17 (link),18 (link))
QPCR, and western blotting were carried out essentially as described previously (10 (link))
Lipids were extracted and sphingolipids quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) (10 (link),19 (link))
Cells were fixed for 5 min in 4% paraformaldehyde in phosphate-buffered saline and blocked by horse serum, and immunocytochemistry was performed using the following primary antibodies: anti-ABCB1 (C219, Abcam, UK), anti-ABCC1 (MRPr1, Monosan, The Netherlands), and anti-SphK1 phospho-Ser225 (ECM Biosciences)

dependent variables

Cell proliferation
Motility
In vitro angiogenesis and lymphangiogenesis
QPCR and western blotting results
Sphingolipid levels
Immunocytochemistry results for ABCB1, ABCC1, and SphK1 phospho-Ser225

control variables

The specificities of anti-ABCC1 and anti-SphK1 antibodies and anti-phospho-SphK1 specific antibody, phospho-Ser225, were previously confirmed using siRNA knockdown (10 (link),20 (no link found))

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!