Cell signaling analysis by flow cytometry

Cell signaling – a process by which cells modulate gene expression in response to extra- and intracellular cues – has become a highly significant part of cell and molecular biology research. Signal transduction inside a cell is often accomplished through a series of reversible modifications of proteins, e.g. phosphorylation, acetylation, myristoylation, glycosylation, and more. Almost all cellular processes – from growth to apoptosis – can be tied to one or more cell signaling pathways. Likewise, most diseases can be linked to irregularities of these pathways, which makes signaling pathway proteins extremely relevant targets for future therapies. 

Antibodies are indispensable for molecular tagging, identification, and analysis via technologies like microscopy, western blot, flow cytometry, and so on. Our recombinantly generated REAfinity™ Antibodies provide several benefits over hybridoma-derived antibody clones, for example, a specifically mutated human IgG1 Fc region that abolishes their binding to FcγRs. This enables more reliable and reproducible flow cytometric analyses of cell signaling. 
 

Embryonic development, which involves careful temporo-spatial coordination of cell proliferation, growth, and differentiation to form highly organized tissues, is a captivating cellular process. In spite of its complexity, only a limited number of signaling pathways take part in embryonic development. These include two types of signaling pathways – juxtacrine signaling, involving cell-to-cell contact via surface proteins (e.g. Notch), and paracrine signaling, involving secreted growth and differentiation factors (e.g. FGF).

Cell-cell and cell-extracellular matrix interactions play crucial roles in diverse processes such as wound healing and cancer invasion. Two classes of cell surface receptors – integrins and cadherins – modulate these interactions, – integrins and cadherins – which organize into large adhesion complexes to influence signaling pathways that mediate cell survival, proliferation, and differentiation.

Cell cycle coordination involves the regulation of its three major phases – mitosis initiation, chromosomal segregation, and cytokinesis. Cell cycle signaling pathways ensure that the cell cycle stages occur in the appropriate order, i.e., one event does not start before the successful completion of the preceding event. BesidesFurthermore, these signaling pathways provide quality control barriers by arresting the process if and when things go wrong.

Signaling pathways involved in cell growth and proliferation fall into four groups: cell cycle regulators, metabolic-anabolic pathway activators, cytoskeleton remodelers, and DNA/replication repair modulators. Based on external stimuli perceived during the G1 phase, cells either decide to proliferate or differentiate. This is achieved by time-dependent coordination between two sets of antagonistic signaling molecules – cyclin-dependent kinases that promote proliferation, and tissue-specific transcription factors that promote differentiation.

Epigenetic modifications, such as DNA methylation, can alter gene expression by adding, removing, or responding to specific epigenetic markers on DNA or histones. DNA methylation, characterized by the addition of a methyl group, are carried out by DNA methyltransferases (DNMTs). DNMTs take part in multiple cellular processes, such as proliferation, embryonic development, and tumor progression.

Both innate and adaptive immune responses involve a complex array of signaling pathways. Macrophages, basophils, neutrophils, NK cells, and others take care of the innate immune response, the first line of defense against pathogens. Adaptive immune response, the second line of defense against pathogens, involves the rapid proliferation of T and B cell lymphocytes. T-cell receptor and B-cell receptor signaling pathways coordinate this response.

The ubiquitin-proteasome system (via the proteasome) and autophagy (via the lysosome) take care of cellular homeostasis by coordinating controlled protein degradation. Proteasomes degrade individual proteins in a highly targeted manner, whereas lysosomes degrade larger cytoplasmic components through autophagy. A multitude of signaling pathways regulate these two processes to achieve cellular homeostasis.

Apoptosis, or programmed cell death, is a highly regulated cellular process for systematic disintegration of cells. There are two major apoptotic signaling pathways: extrinsic and intrinsic. The extrinsic pathway, activated by extracellular ligands, involves cell surface death receptors leading to the formation of death-inducing signaling complexes. The intrinsic pathway, activated by intracellular signals, involves the mitochondria leading to the formation of apoptosomes. 

Dysregulation of several signaling pathways involved in different cellular processes have also been associated with cancer. Mutation or overexpression of proteins in these signaling pathways are commonly observed in cancer. Hence, signaling pathway proteins are key targets in cancer research and drug discovery.