Volume 16 Issue 11, November 2021

This Protocol Extension discusses several approaches to analyzing clonogenic growth of mammalian cells in vitro, using a modular framework to facilitate the use of various formats to fully optimize clonogenic growth.

This protocol describes a scalable approach for joint profiling of chromatin accessibility and gene expression in single cells or nuclei that relies on multiple rounds of combinatorial indexing and can identify cell-specific regulatory elements.

This protocol explains how to use and apply COMETS (computation of microbial ecosystems in time and space), which extends dynamic flux balance analysis to generate simulations of multiple microbial species in molecularly complex and spatially structured environments.

Protein lipidation is challenging to analyze on the proteome level. This protocol describes metabolic incorporation of lipid probes and their capture via click chemistry, allowing in-gel fluorescence visualization or mass spectrometry analysis.

Dysregulated growth and proliferation of tumors is related to metabolic changes. This protocol assesses metabolism in intact tumors using stable isotope-labeled nutrients delivered via bolus injection and continuous infusion in mice and humans.

This protocol describes a procedure for stabilizing and characterizing a transient high-energy RNA structure called the excited state, using relaxation dispersion NMR spectroscopy.

The polarity of gastrointestinal organoids is reversed to study epithelial biology and host–microbe interactions. Access to the apical surface of the epithelium is increased while preserving epithelial integrity and secretory and absorptive functions.

The newly transcribed RNA-bound proteome (or RNA interactome) is captured using this protocol by time-dependent incorporation of 5-ethynyluridine into newly transcribed RNAs followed by UV-mediated RNA-protein cross-linking, click-chemistry-aided biotinylation and affinity isolation of RNA–protein complexes.

The authors present a magnetic purification protocol that separates scarce macropinosomes from other endocytic vesicles at a high purity and in an unbiased manner for label-free quantitative mass spectrometry.

The solution behavior of drugs is determined by NMR, enabling the presence of fast-tumbling lone drug molecules, small drug aggregates and slow-tumbling colloids to be determined.

Mota et al. describe a protocol for the rapid, room-temperature, kit-based synthesis of ¹⁸F-FDS for positron emission tomography imaging.

Split-GFP-based contact site sensors (SPLICS) are capable of reconstituting fluorescence when two opposing membranes come into close proximity. SPLICS can be used to quantify membrane contact sites between organelles in both fixed and living cells.

Quantitative MRI data acquired from patients with locally advanced breast cancer are used to calibrate a biophysical, reaction–diffusion mathematical model to predict response to neoadjuvant therapy on an individual patient basis.

The authors present a protocol for expressing and biophysically characterizing rationally engineered SARS-CoV-2 spike proteins in Freestyle 293 and ExpiCHO cell lines.