What is multiplexing?
Multiplexing, in biological applications, is defined by the simultaneous evaluation of several experimental elements, thereby increasing the throughput of analysis and reducing the burden of time and cost associated with the investigation of those individual components. [1-3] Increasingly, researchers are combining various assays to interrogate complex biological questions related to gene expression profiles, signaling pathway events, and cell health and proliferation indicators, to name a few. [4-8] Through advanced technologies, using flow cytometry, imaging, plate reader, and mass spectrometry formats, multiparameter analysis of a given sample, at the single cell level, is made possible. [6-11] Further coupling of technologies, as with RNA detection through branched DNA amplification and immunolabeling, or with acoustic cytometry and distinct fluorescence emitting probes, will continue to expand the number of parameters that can be analyzed concomitantly. This review highlights a few multiplexed applications developed by Thermo Fisher Scientific, to help biomedical researchers overcome many of the limitations caused by sample scarcity, or variability across samples and platforms. Navigate to the related BioProbes articles for a more detailed discussion around each topic area.
Simultaneous visualization of protein and RNA expression at the single-cell level
Invitrogen PrimeFlow RNA and ViewRNA Cell Plus are two assays that effectively combine various technologies to study RNA molecules on a single-cell level. Compatible with the Cellnsight CX7 High-Content Screening platform, the ViewRNA Cell Plus assay combines ViewRNA ISH technology, a proprietary fluorescent in situ hybridization (FISH) and branched DNA (bDNA) amplification technique, with antibody-based protein detection to simultaneously visualize RNA and protein in single cells. ViewRNA Cell Plus assay made traditional incompatible immunocytochemistry (ICC) and in situ hybridization (ISH) compatible. Similarly, the PrimeFlow RNA assay employs FISH with bDNA signal amplification for the simultaneous detection of up to four RNA targets. This can be used in combination with immunolabeling for both cell-surface and intracellular proteins detection using fluorophore-conjugated antibodies for analysis by flow cytometry.
Interested in learning more? Read these BioProbes articles!
BioProbes 75: Evaluate both RNA and protein targets in single cells
Robust cell health analysis using multiplexable cell viability assays
Viability assessment is critical in many application areas in basic, pre-clinical and clinical research settings. Many physiological or morphological parameters need to be understood, at the subcellular level, to determine cell health. Viability probes can be multiplexed with a combination of different measurements to provide more cellular information than any single-parameter assay. For instance, Figure 3 demonstrates a workflow to investigate both proliferation and viability by using CyQUANT Direct cell proliferation assay and PrestoBlue viability reagent. CyQUANT direct is a highly sensitive DNA-content based assay and PrestoBlue cell viability reagent is a metabolism-based assay. The use of the PrestoBlue and CyQUANT Direct assays together allow for cell metabolism, DNA content, and changes in membrane permeability, to be assessed in the same sample, using distinct fluorescent signals. The tandem CyQUANT Direct and PrestoBlue protocol is easy to carry out and requires no wash or cell lysis steps.
Discover how! BioProbes 73: CyQUANT direct and PrestoBlue viability assays work together
Explore ways to cooperatively investigate mitochondrial morphology and function
Monitoring changes in mitochondrial morphology and function are valuable indicators of cell health. Mitochondrial morphology reagents, that allow for the assessment of membrane integrity, can be combined with functional probes to provide more information about mitochondrial health. Invitrogen reagents can be used to study mitochondrial membrane potential, calcium flux, oxidative phosphorylation, autophagy/mitophagy and cytosolic pH. Furthermore, improvements in the development of probes has enabled researchers with more fluorescence options that can be combined to study mitochondria. For example, in Figure 4 below, Invitrogen CellLight Mitochondria-GFP or CellLight Mitochondria-RFP can be combined with dyes such as Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) to monitor mitochondrial structural integrity while also assessing membrane potential.
Read more about mitochondrial health!
BioProbes 72: Tools to study mitochondrial morphology and function
Multiplexable assays for in situ apoptosis detection in combination with multiple fluorescent dyes
Changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and fragmentation of cellular DNA are all indicators of late stage apoptosis. Detected is made possible using DNA-binding dyes and in situ hybridization. Invitrogen Click-iT Plus, with terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assays, enables researchers to achieve specific and sensitive DNA fragmentation detection that can be multiplexed with other fluorescence-based approaches. The Click-iT Plus TUNEL assay offers gentle reaction and highly sensitive detection conditions that can be applied to wide range of cell types, while including various fluorescent proteins or reagents. Figure 5 below demonstrates the combinatorial of Click-iTTM Plus TUNEL Assay with Alexa FluorTM 594 dye, muscularis externa expressing GFP, nuclei labeled with Thermo Scientific Hoechst 33342 dye, and actin staining with Invitrogen Alexa FluorTM 647 phalloidin in a single tissue section analyzed by microscopy.
Find out more!
- Krutzik PO, et.al., Fluorescent cell barcoding for multiplex flow cytometry. Curr Protoc Cytom 2011; Chapter:Unit6.31.
- Bradford JA, et.al., Fluorescence-intensity multiplexing: Simultaneous seven-marker, two-color immunophenotyping using flow cytometry. Cytometry A 2004; 61A:142–152.
- Smurthwaite C, et.al. Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry. Cytometry A. 2013; 85A:105-113.
- Grant GD, et.al., Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control. Mol Biol Cell 2012; 23:3079–3093.
- Lu R, et.al., Tracking single hematopoietic stem cells in-vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat Biotechnol 2011; 29:928–933.
- Krutzik PO, Nolan GP. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 2006; 3:361–368.
- Edwards BS, et.al.,. High-throughput cytotoxicity screening by propidium iodide staining. Curr Protoc Cytom 2007; Chapter 9:Unit9.24.
- Violin JD, et.al., A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 2003; 161:899–909.
- Ashcroft RG, Lopez PA. Commercial high-speed machines open new opportunities in high throughput flow cytometry (HTFC). J Immunol Methods 2000; 243:13–24.
- Edwards BS, et.al., High-throughput flow cytometry for drug discovery. Expert Opin Drug Discov 2007; 2:685–696
- Barteneva NS, et.al., Imaging flow cytometry. Coping with heterogeneity in biological systems. J Histochem Cytochem. 20012; Chapter 60:Unit10:723-733.