Experimental Methods

In addition to performing established biochemical and cell biological assays, the HMS LINCS Center participates in the development and improvement of cutting-edge experimental methods. The goal of the Center is to integrate these new methods into our adaptive data collection approach. The following list of methods developed by HMS LINCS will be updated regularly as our work progresses.

 

CycIF, a high-throughput cyclic immunofluorescence method

Nature Communications (2015) [vol TBD], [page TBD].
doi:TBD / PMID:TBD / PMC:TBD

Lin et al (2015)There is an emerging need in numerous fields of biological research to increase the multiplicity of single-cell measurements. Multiplexed single-cell techniques have the potential to reveal important interdependencies between a cell’s local environment and its differentiation status, signal transduction pathway activity, and morphological phenotypes that are not evident when each feature is measured independently. To this end, we developed a robust, multiplexed immunofluorescence imaging method, named Cyclic Immunofluorescence (CycIF), using public domain chemistry and existing instruments that enables low-cost, high-dimensionality imaging assays at the single-cell level.

 

A one-step imaging assay scores mitotic arrest and apoptotic state in live cells

J Biomol Screen (2013) 18 (9), 1062–1071.
doi:10.1177/1087057113493804 / PMID:23788527 / PMCID:PMC3783590

Tang et al (2013)Discriminating different mechanisms that compromise drug sensitivity in cells in culture requires multiplexed readout of response, which is often accomplished using mRNA profiling, multiplexed gene expression reporters, and high-content imaging assays. These assays can be highly informative but typically are costly and complex. Furthermore, it can be difficult to infer alternative mechanistic effects on drug response pathways from gene expression and other multiplex readouts where the relationship between readout and drug response pathway is complex. Here, we describe a new one-step, no-wash imaging assay that uses three dyes (Hoechst33342, LysoTracker-Red, and DEVD-NucView488) to stain living cells and enables measurement of multiple physiological changes in cells related to mitotic and apoptotic status following treatment with anti-mitotic small molecule drugs.

McAllister et al (2013)

ActivX ATP probe-enhanced, high-throughput
mass spectrometry

Analytical Chemistry (2013) 85 (9), 4666–4674.
doi:10.1021/ac303478g / PMID:23607489 / PMCID:PMC3771683

Kinome-wide profiling aims to provide a systematic, unbiased look at kinase levels and modification states across biological and pathological processes. Current kinase analysis methods, however, are low in throughput and often poor at detecting low-abundance kinases unless time-consuming cellular fractionation steps are undertaken. We describe here a robust method that combines ActivX ATP probe (AAP) affinity reagents (ATP analogues) with 6-plex tandem mass tag (TMT) isotopic labeling of cell lysates and enables multiplexed analysis of ~90 kinases across six conditions in a single LC-MS run.