An official website of the United States government
Abstract Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS‐based method, termed Two‐Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of live cells expressing protein constructs Basic Protocol 2: Image acquisition and noise correction Basic Protocol 3: Drawing and segmenting regions of interest Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments Basic Protocol 5: Combining data subsets using a manual procedure (Optional) Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5) Basic Protocol 6: Performing meta‐analysis of brightness spectrograms Alternate Protocol 2: Performing meta‐analysis of brightness spectrograms (alternative to Basic Protocol 6) Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional) Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7) Support Protocol: Monomeric brightness determination
more »
« less
Measuring Cytoskeletal Mechanical Fluctuations and Rheology with Active Micropost Arrays
Abstract The dynamics of the cellular actomyosin cytoskeleton are crucial to many aspects of cellular function. Here, we describe techniques that employ active micropost array detectors (AMPADs) to measure cytoskeletal rheology and mechanical force fluctuations. The AMPADS are arrays of flexible poly(dimethylsiloxane) (PDMS) microposts with magnetic nanowires embedded in a subset of microposts to enable actuation of those posts via an externally applied magnetic field. Techniques are described to track the magnetic microposts’ motion with nanometer precision at up to 100 video frames per second to measure the local cellular rheology at well‐defined positions. Application of these high‐precision tracking techniques to the full array of microposts in contact with a cell also enables mapping of the cytoskeletal mechanical fluctuation dynamics with high spatial and temporal resolution. This article describes (1) the fabrication of magnetic micropost arrays, (2) measurement protocols for both local rheology and cytoskeletal force fluctuation mapping, and (3) special‐purpose software routines to reduce and analyze these data. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fabrication of magnetic micropost arrays Basic Protocol 2: Data acquisition for cellular force fluctuations on non‐magnetic micropost arrays Basic Protocol 3: Data acquisition for local cellular rheology measurements with magnetic microposts Basic Protocol 4: Data reduction: determining microposts’ motion Basic Protocol 5: Data analysis: determining local rheology from magnetic microposts Basic Protocol 6: Data analysis for force fluctuation measurements Support Protocol 1: Fabrication of magnetic Ni nanowires by electrodeposition Support Protocol 2: Configuring Streampix for magnetic rheology measurements
more »
« less
- PAR ID:
- 10381140
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 2
- Issue:
- 5
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Indentation testing is the most common approach to quantify mechanical brain tissue properties. Despite a myriad of studies conducted already, reported stiffness values vary extensively and continue to be subject of study. Moreover, the growing interest in the relationship between the brain's spatially heterogeneous microstructure and local tissue stiffness warrants the development of standardized measurement protocols to enable comparability between studies and assess repeatability of reported data. Here, we present three individual protocols that outline (1) sample preparation of a 1000‐µm thick coronal slice, (2) a comprehensive list of experimental parameters associated with the FemtoTools FT‐MTA03 Micromechanical Testing System for spherical indentation, and (3) two different approaches to derive the elastic modulus from raw force‐displacement data. Lastly, we demonstrate that our protocols deliver a robust experimental framework that enables us to determine the spatially heterogeneous microstructural properties of (mouse) brain tissue. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Mouse brain sample preparation Basic Protocol 2: Indentation testing of mouse brain tissue using the FemtoTools FT‐MTA03 Micromechanical Testing and Assembly System Basic Protocol 3: Tissue stiffness identification from force‐displacement datamore » « less
-
Abstract Accurately mapping changes in cellular membrane potential across large groups of neurons is crucial for understanding the organization and maintenance of neural circuits. Measuring cellular voltage changes by optical means allows greater spatial resolution than traditional electrophysiology methods and is adaptable to high‐throughput imaging experiments. VoltageFluors, a class of voltage‐sensitive dyes, have recently been used to optically study the spontaneous activity of many neurons simultaneously in dissociated culture. VoltageFluors are particularly useful for experiments investigating differences in excitability and connectivity between neurons at different stages of development and in different disease models. The protocols in this article describe general procedures for preparing dissociated cultures, imaging spontaneous activity in dissociated cultures with VoltageFluors, and analyzing optical spontaneous activity data. © 2021 Wiley Periodicals LLC. This article was corrected on 20 July 2022. See the end of the full text for details. Basic Protocol 1: Preparation of dissociated rat hippocampal or cortical cultures Alternate Protocol: Preparation of microisland dissociated cultures Basic Protocol 2: Imaging of spontaneous activity in dissociated cultures using voltage‐sensitive dyes Basic Protocol 3: Analysis of spontaneous activity imaging datamore » « less
-
Abstract Cells in living tissues are exposed to substantial mechanical forces and constraints imposed by neighboring cells, the extracellular matrix, and external factors. Mechanical forces and physical confinement can drive various cellular responses, including changes in gene expression, cell growth, differentiation, and migration, all of which have important implications in physiological and pathological processes, such as immune cell migration or cancer metastasis. Previous studies have shown that nuclear deformation induced by 3D confinement promotes cell contractility but can also cause DNA damage and changes in chromatin organization, thereby motivating further studies in nuclear mechanobiology. In this protocol, we present a custom‐developed, easy‐to‐use, robust, and low‐cost approach to induce precisely defined physical confinement on cells using agarose pads with micropillars and externally applied weights. We validated the device by confirming nuclear deformation, changes in nuclear area, and cell viability after confinement. The device is suitable for short‐ and long‐term confinement studies and compatible with imaging of both live and fixed samples, thus presenting a versatile approach to studying the impact of 3D cell confinement and nuclear deformation on cellular function. This article contains detailed protocols for the fabrication and use of the confinement device, including live cell imaging and labeling of fixed cells for subsequent analysis. These protocols can be amended for specific applications. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Design and fabrication of the confinement device wafer Basic Protocol 2: Cell confinement assay Support Protocol 1: Fixation and staining of cells after confinement Support protocol 2: Live/dead staining of cells during confinementmore » « less
-
Abstract Multiple sequence alignments and phylogenetic trees are rich in biological information and are fundamental to research in biology. PhyKIT is a tool for processing and analyzing the information content of multiple sequence alignments and phylogenetic trees. Here, we describe how to use PhyKIT for diverse analyses, including (i) constructing a phylogenomic supermatrix, (ii) detecting errors in orthology inference, (iii) quantifying biases in phylogenomic data sets, (iv) identifying radiation events or lack of resolution using gene support frequencies, and (v) conducting evolution‐based screens to facilitate gene function prediction. Several PhyKIT functions that streamline multiple sequence alignment and phylogenetic processing—such as renaming FASTA entries or tree tips—are also discussed. These protocols demonstrate how simple command‐line operations in the unified framework of PhyKIT facilitate diverse phylogenomic data analysis and processing, from supermatrix construction and diagnosis to gaining clues about gene function. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Installing PhyKIT and syntax for usage Basic Protocol 2: Constructing a phylogenomic supermatrix Basic Protocol 3: Detecting anomalies in orthology relationships Basic Protocol 4: Quantifying biases in phylogenomic data matrices and related measures Basic Protocol 5: Identifying polytomies Basic Protocol 6: Assessing gene‐gene coevolution as a genetic screenmore » « less
