Analysis Of Single-cell Responses Research Articles

An Integrated Preprocessing Approach for Exploring Single-Cell Gene Expression in Rare Cells

Exploring the variability in gene expressions of rare cells at the single-cell level is critical for understanding mechanisms of differentiation in tissue function and development as well as for disease diagnostics and cancer treatment. Such studies, however, have been hindered by major difficulties in tracking the identity of individual cells. We present an approach that combines single-cell picking, lysing, reverse transcription and digital polymerase chain reaction to enable the isolation, tracking and gene expression analysis of rare cells. The approach utilizes a photocleavage bead-based microfluidic device to synthesize and deliver stable cDNA for downstream gene expression analysis, thereby allowing chip-based integration of multiple reactions and facilitating the minimization of sample loss or contamination. The utility of the approach was demonstrated with QuantStudio digital PCR by analyzing the radiation and bystander effect on individual IMR90 human lung fibroblasts. Expression levels of the Cyclin-dependent kinase inhibitor 1a (CDKN1A), Growth/differentiation factor 15 (GDF15), and Prostaglandin-endoperoxide synthase 2 (PTGS2) genes, previously shown to have different responses to direct and bystander irradiation, were measured across individual control, microbeam-irradiated or bystander IMR90 cells. In addition to the confirmation of accurate tracking of cell treatments through the system and efficient analysis of single-cell responses, the results enable comparison of activation levels of different genes and provide insight into signaling pathways within individual cells.

The use of quantitative imaging to investigate regulators of membrane trafficking in Arabidopsis stomatal closure.

Expansion of gene families facilitates robustness and evolvability of biological processes but impedes functional genetic dissection of signalling pathways. To address this, quantitative analysis of single cell responses can help characterize the redundancy within gene families. We developed high-throughput quantitative imaging of stomatal closure, a response of plant guard cells, and performed a reverse genetic screen in a group of Arabidopsis mutants to five stimuli. Focussing on the intersection between guard cell signalling and the endomembrane system, we identified eight clusters based on the mutant stomatal responses. Mutants generally affected in stomatal closure were mostly in genes encoding SNARE and SCAMP membrane regulators. By contrast, mutants in RAB5 GTPase genes played specific roles in stomatal closure to microbial but not drought stress. Together with timed quantitative imaging of endosomes revealing sequential patterns in FLS2 trafficking, our imaging pipeline can resolve non-redundant functions of the RAB5 GTPase gene family. Finally, we provide a valuable image-based tool to dissect guard cell responses and outline a genetic framework of stomatal closure.

T Cell Dynamic Activation and Functional Analysis in Nanoliter Droplet Microarray.

ObjectiveCharacterization of the heterogeneity in immune reactions requires assessing dynamic single cell responses as well as interactions between the various immune cell subsets. Maturation and activation of effector cells is regulated by cell contact-dependent and soluble factor-mediated paracrine signalling. Currently there are few methods available that allow dynamic investigation of both processes simultaneously without physically constraining non-adherent cells and eliminating crosstalk from neighboring cell pairs. We describe here a microfluidic droplet microarray platform that permits rapid functional analysis of single cell responses and co-encapsulation of heterotypic cell pairs, thereby allowing us to evaluate the dynamic activation state of primary T cells.MethodsThe microfluidic droplet platform enables generation and docking of monodisperse nanoliter volume (0.523 nl) droplets, with the capacity of monitoring a thousand droplets per experiment. Single human T cells were encapsulated in droplets and stimulated on-chip with the calcium ionophore ionomycin. T cells were also co-encapsulated with dendritic cells activated by ovalbumin peptide, followed by dynamic calcium signal monitoring.ResultsIonomycin-stimulated cells depicted fluctuation in calcium signalling compared to control. Both cell populations demonstrated marked heterogeneity in responses. Calcium signalling was observed in T cells immediately following contact with DCs, suggesting an early activation signal. T cells further showed non-contact mediated increase in calcium level, although this response was delayed compared to contact-mediated signals.ConclusionsOur results suggest that this nanoliter droplet array-based microfluidic platform is a promising technique for assessment of heterogeneity in various types of cellular responses, detection of early/delayed signalling events and live cell phenotyping of immune cells.

Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors.

Acquired drug resistance is a key factor in the failure of chemotherapy. Due to intratumoral heterogeneity, cancer cells depict variations in intracellular drug uptake and efflux at the single cell level, which may not be detectable in bulk assays. In this study we present a droplet microfluidics-based approach to assess the dynamics of drug uptake, efflux and cytotoxicity in drug-sensitive and drug-resistant breast cancer cells. An integrated droplet generation and docking microarray was utilized to encapsulate single cells as well as homotypic cell aggregates. Drug-sensitive cells showed greater death in the presence or absence of Doxorubicin (Dox) compared to the drug-resistant cells. We observed heterogeneous Dox uptake in individual drug-sensitive cells while the drug-resistant cells showed uniformly low uptake and retention. Dox-resistant cells were classified into distinct subsets based on their efflux properties. Cells that showed longer retention of extracellular reagents also demonstrated maximal death. We further observed homotypic fusion of both cell types in droplets, which resulted in increased cell survival in the presence of high doses of Dox. Our results establish the applicability of this microfluidic platform for quantitative drug screening in single cells and multicellular interactions.

Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression.

Methods for analysis of T cell function have traditionally relied upon measurements of proliferation or cytokine expression in bulk cultures of PBMC in long term incubations with polyclonal mitogens or putative antigen. These techniques suffer from the drawback that they do not enable analysis of single cell responses in the context of unselected cellular backgrounds. In addition these methods are not sensitive enough to rapidly assess rare event responses characteristic of cognate memory T cell responses. This review discusses recently developed flow cytometric methods designed to rapidly assess leukocyte subset cytokine responses to polyclonal activators and specific antigen in PBMC and whole blood samples. These procedures determine the percentages of activated cells and the identification of leucocyte subsets capable of expressing various cytokines and cell surface antigens. The ability to assess key intracellular functional markers by multiparameter flow cytometry offers some unique advantages in a number of clinical applications. The technical simplicity and rapidity of the flow cytometric intracellular cytokine detection techniques described in this report, as well as the widespread availability of appropriate flow cytometers and cell surface directed antibodies in clinical laboratories, suggests the possibility that this technique could be broadly applicable to the clinical evaluation of immune status. Since any cell type can be identified with this approach, responses to a variety of clinically relevant stimuli in virtually any leukocyte subset can be evaluated including monocyte responses to LPS, and T cell responses to mitogens and a variety of bacterial and viral antigens. The significance of measuring low frequency antigen-specific responses with respect to clinical significance in assessing immune status in a variety of clinical conditions and determining efficacy or immunotoxicity of drugs and vaccine antigens is discussed.

Rapid assessment of antigen induced cytokine expression in memory T cells by flow cytometry

Methods for analysis of T cell responses to specific antigen have traditionally relied on measurements of proliferation or cytokine expression in bulk cultures of PBMC in long term incubations with putative antigen. These techniques suffer from the drawback that they do not enable analysis of single cell responses in the context of unselected cellular backgrounds. It is increasingly important to not only identify cells on the basis of expression of unique surface antigens but also to determine functional and molecular parameters of individual cells in response to a variety of stimuli. We have recently developed methodologies to rapidly assess T-cell subset responses to polyclonal activators and specific antigen in whole blood. These procedures determine the percentages of activated cells and the identification of leucocyte subsets capable of expressing various cytokines and cell surface antigens. Multiparameter analysis of CD69+/cytokine+expression in T cells in response to specific antigen (e.g. CMV, mumps) demonstrated a range of frequencies from 0.05% to 5.0% within 6 h. Frequencies of responding T cells were consistent but varied depending upon the antigen. Antigen specific T cell responses were host specific and both positively and negatively regulated by antibodies to co-receptors involved in APC-T cell interactions. These technologies will be discussed in terms of application to problems of measuring immune function parameters in disease. The modified technique described in this report is compatible with simple and rapid analysis of clinical samples and provides a means to directly examine the effects of in vivo drug concentrations on T cell immunity. Studies are in progress to examine the sensitivity of this cellular assay to drugs and other therapeutic modalities in clinical samples.

A Possible Explanation for the Effect of Acupuncture on Pain

The physiological basis for the interruption of pain by acupuncture for anesthesia and analgesia has been of great interest in the last few years, since communications with Red China have become more common. The physiological basis for the effect of acupuncture on pain is still conjectural, but may well be related to a peripheral field effect from differential sensory input. To study this phenomenon, twelve cats were used in an acute preparation. Responses to C fiber input were recorded in the medial thalamus, both by average evoked responses and by single cell recordings with microelectrodes. When large myelinated fiber input from a different portion of the body was used in conjunction with noxious input, gross evoked responses to pain were clearly diminished, and single cell responses were altered. This modulation of C fiber input by large fibers was long lasting – a single interaction between two inputs led to a diminution of evoked responses to the noxious stimulus alone lasting up to 1 h. Analysis of single cell responses to noxious and non-noxious input to the thalamus suggested that a supraspinal gating mechanism was responsible for the different types of cellular responses being recorded in the centre median pulvinar and parafascicularis area. This supraspinal gating is discussed in relation to the analgesic effect of acupuncture nerve stimulation and other methods of pain relief as well as the theoretical method of processing sensory information in the thalamus.