Basic Techniques in Mammalian Cell Tissue Culture

Phelan, Katy; May, Kristin M.

doi:10.1002/0471143030.cb0101s66

Cited by 131 publications

(148 citation statements)

References 12 publications

(13 reference statements)

Supporting

Mentioning

145

Contrasting

Unclassified

Order By: Relevance

“…Additional reagents and equipment for basic cell culture techniques including determining cell viability by trypan blue exclusion ( unit 1.1 ; Phelan and May, 2015), and fluorescence-activated cell sorting (Robinson et al, 2015)…”

Section: Support Protocolmentioning

confidence: 99%

“…The next morning: Pre-equilibrate 25 ml of fully supplemented CHOs medium (see step 1) in a 125-ml conical flask in a 37°C, 5% CO 2 incubator for 1 hr.During this time, pool the cells from all wells and determine their viability using standard methods of choice [e.g., trypan blue exclusion; see unit 1.1 (Phelan and May, 2015); the viability should be > 90%]. Transfer the cells into the flask and place into the shaking incubator.…”

Section: Support Protocolmentioning

confidence: 99%

“…Split the cells into separate flasks if the volume grows to greater than 50 ml, with a minimum volume of 25 ml per flask. When 100 ml of volume has been achieved, transfer the culture to a 250-ml shaking flask. unit 1.1 (Phelan and May, 2015) includes protocols for basic cell culture.…”

Section: Support Protocolmentioning

confidence: 99%

“…During this time, pool the cells from all wells and determine their viability using standard methods of choice [e.g., trypan blue exclusion; see unit 1.1 (Phelan and May, 2015); the viability should be > 90%]. Transfer the cells into the flask and place into the shaking incubator.…”

Section: Support Protocolmentioning

confidence: 99%

“…Every 48 hr, determine the viable number of cells ( unit 1.1 ; Phelan and May, 2015), allowing the culture to expand to 2–3 × 10 6 cells/ml.

During each passage, filter the cells through a 50-μm nylon mesh to remove the clumps.

…”

Section: Support Protocolmentioning

confidence: 99%

See 4 more Smart Citations

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

et al. 2015

View full text Add to dashboard Cite

Glass‐supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell‐cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene‐expression analysis. Finally, we describe how to design and prepare transmembrane‐anchored protein‐laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane‐anchored proteins may have many novel applications in cell biology and immunology. © 2015 by John Wiley & Sons, Inc.

show abstract

Section: Support Protocolmentioning

confidence: 99%

Section: Support Protocolmentioning

confidence: 99%

Section: Support Protocolmentioning

confidence: 99%

Section: Support Protocolmentioning

confidence: 99%

“…Every 48 hr, determine the viable number of cells ( unit 1.1 ; Phelan and May, 2015), allowing the culture to expand to 2–3 × 10 6 cells/ml.

During each passage, filter the cells through a 50-μm nylon mesh to remove the clumps.

…”

Section: Support Protocolmentioning

confidence: 99%

See 3 more Smart Citations

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

et al. 2015

View full text Add to dashboard Cite

show abstract

Immunofluorescence Staining

Donaldson

2015

CP Cell Biology

View full text Add to dashboard Cite

This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to a fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescence microscope. This unit describes the widely used and powerful technique of localization of proteins in cells by immunofluorescence. The location can be determined by double labeling with an antibody directed against a protein of known location. The technique can be used as a supplement to immunolocalization by electron microscopy and subcellular fractionation. It allows not only identification of the antigen distribution in the cell but also a survey of the dynamic aspects of protein movements in the cell—on and off membranes, into and out of the nucleus, and through membrane traffic pathways. © 2015 by John Wiley & Sons, Inc.

show abstract

Three‐Dimensional Patterning of the ECM Microenvironment Using Magnetic Nanoparticle Self Assembly

Kim

Tanner

2016

CP Cell Biology

View full text Add to dashboard Cite

This protocol describes a way to introduce topography to three‐dimensional (3D) biomaterials. The self‐assembling behavior of magnetic particles can be exploited to form nanoscale to microscale fibers, such that one can dissect the contribution of topography on cell behavior, which is independent of other physical properties of the biomaterial (e.g., stiffness). The magnetic particles are chemically cross‐linked with several extracellular matrix (ECM) proteins and then using magnetic force‐mediated assembly, one can program aligned nanofibers in a 3D hydrogel. This process allows the creation of diverse topographic patterns in 3D, including isotropic, anisotropic (fibril), or interfaced architectures, without changing the bulk stiffness of the scaffold material. This anisotropic architecture guides the dendritic protrusions of cells, which can be compared to cells grown in an isotropic architecture lacking spatial guidance cues. Several cell types, such as fibroblasts and neurons, have been cultured in this engineered 3D matrix. This technology provides an easy way to construct nano‐bio interfaces for various biomedical engineering applications as well as dissect the role of topography in various cell behaviors. © 2016 by John Wiley & Sons, Inc.

show abstract

Basic Techniques in Mammalian Cell Tissue Culture

Cited by 131 publications

References 12 publications

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

Supported Lipid Bilayer Technology for the Study of Cellular Interfaces

Immunofluorescence Staining

Three‐Dimensional Patterning of the ECM Microenvironment Using Magnetic Nanoparticle Self Assembly

Contact Info

Product

Resources

About