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Cell Biology

Categories
+ Cell engineering
+ Cell imaging
+ Cell isolation and culture
+ Cell metabolism
+ Cell movement
- Cell signaling
Autophagy
Development
Intracellular Signaling
Phosphorylation
Quorum sensing
Respiration
Second messenger
Stress response
Synaptic transmision
+ Cell staining
+ Cell structure
+ Cell Transplantation
+ Cell viability
+ Cell-based analysis
+ Model organism culture
+ Organelle isolation
+ Single cell analysis
+ Tissue analysis
Protocols in Past Issues

Analysis of Guard Cell Readouts Using Arabidopsis thaliana Isolated Epidermal Peels
RP Rosario Pantaleno
PS Paula Schiel
CG Carlos García-Mata
DS Denise Scuffi
Q&A 0 Q&A
2385 Views
Jul 20, 2024

Stomata are pores surrounded by a pair of specialized cells, called guard cells, that play a central role in plant physiology through the regulation of gas exchange between plants and the environment. Guard cells have features like cell-autonomous responses and easily measurable readouts that have turned them into a model system to study signal transduction mechanisms in plants. Here, we provide a detailed protocol to analyze different physiological responses specifically in guard cells. We describe, in detail, the steps and conditions to isolate epidermal peels with tweezers and to analyze i) stomatal aperture in response to different stimuli, ii) cytosolic parameters such as hydrogen peroxide (H2O2), glutathione redox potential (EGSH), and MgATP-2 in vivo dynamics using fluorescent biosensors, and iii) gene expression in guard cell–enriched samples. The importance of this protocol lies in the fact that most living cells on epidermal peels are guard cells, enabling the preparation of guard cell–enriched samples.

FRET Reporter Assays for cAMP and Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in Living Cells
BM Brandon T. Milliken
Robert P. Doyle Robert P. Doyle
George G. Holz George G. Holz
OC Oleg G. Chepurny
Q&A 0 Q&A
6926 Views
Jun 5, 2020
Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca2+. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors H188 and YC3.60 so that it is possible to monitor the kinetics with which alterations of [cAMP] or [Ca2+] occur in monolayers or suspensions of living cells exposed to GPCR agonists. This protocol uses HEK293 cell lines doubly transfected with a FRET biosensor and a recombinant GPCR of interest (e.g., glucagon receptors, CCK2 receptors, or NPY2R receptors). The protocol allows for rapid screening of small molecule GPCR agonists and antagonists, and it is also useful for discovery of synthetic mono-, dual-, and tri- agonist peptides with GPCR activating properties.

Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System
OY O. Sascha Yousefi
MH Maximilian Hörner
MW Maximilian Wess
VI Vincent Idstein
WW Wilfried Weber
WS Wolfgang W. A. Schamel
Q&A 0 Q&A
5863 Views
Mar 5, 2020
T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx.

Endpoint or Kinetic Measurement of Hydrogen Sulfide Production Capacity in Tissue Extracts
CH Christopher Hine
JM James R. Mitchell
Q&A 0 Q&A
11547 Views
Jul 5, 2017
Hydrogen sulfide (H2S) gas is produced in cells and tissues via various enzymatic processes. H2S is an important signaling molecule in numerous biological processes, and deficiencies in endogenous H2S production are linked to cardiovascular and other health complications. Quantitation of steady-state H2S levels is challenging due to volatility of the gas and the need for specialized equipment. However, the capacity of an organ or tissue extract to produce H2S under optimized reaction conditions can be measured by a number of current assays that vary in sensitivity, specificity and throughput capacity. We developed a rapid, inexpensive, specific and relatively high-throughput method for quantitative detection of H2S production capacity from biological tissues. H2S released into the head space above a biological sample reacts with lead acetate to form lead sulfide, which is measured on a continuous basis using a plate reader or as an endpoint assay.

cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer)

CK Cassandra Koole
DW Denise Wootten
PS Patrick Sexton
Q&A 0 Q&A
13620 Views
Jul 5, 2013
Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated Gs protein-coupled receptors. Conversely, formation of cAMP is inhibited by Gi protein-coupled receptors. This protocol has been optimized for the detection of ligand-mediated cAMP accumulation in adherent immortal cell lines expressing Gs-coupled receptors.