220-plex microRNA expression profile of a single cell

Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.     

Development and optimization of a metabolomic method for analysis of adherent cell cultures

In this investigation, a gas chromatography/mass spectrometry (GC/MS)-based metabolomic protocol for adherent cell cultures was developed using statistical design of experiments. Cell disruption, metabolite extraction, and the GC/MS settings were optimized aiming at a gentle, unbiased, sensitive, and high-throughput metabolomic protocol. Due to the heterogeneity of the metabolome and the inherent selectivity of all analytical techniques, development of unbiased protocols is highly complex. Changing one parameter of the protocol may change the response of many groups of metabolites. In this investigation, statistical design of experiments and multivariate analysis also allowed such interaction effects to be taken into account. The protocol was validated with respect to linear range, precision, and limit of detection in a clonal rat insulinoma cell line (INS-1 832/13). The protocol allowed high-throughput profiling of metabolites covering the major metabolic pathways. The majority of metabolites displayed a linear range from a single well in a 96-well plate up to a 10 cm culture dish. The method allowed a total of 47 analyses to be performed in 24 h.     

Single Cell Ganglioside Catabolism in Primary Cerebellar Neurons and Glia

Cell-to-cell heterogeneity in ganglioside catabolism was determined by profiling fluorescent tetramethylrhodamine-labeled GM1 (TMR-GM1) breakdown in individual primary neurons and glia from the rat cerebellum. Cells isolated from 5 to 6 day old rat cerebella were cultured for 7 days, and then incubated for 14 h with TMR-GM1. Intact cells were recovered from cultures by mild proteolysis, paraformaldehyde fixed, and subjected to single cell analysis. Individual cells were captured in a capillary, lysed, and the released single-cell contents analyzed by capillary electrophoresis with quantitative laser-induced fluorescent detection of metabolites. Non-neuronal cells on average took up much more exogenous TMR-GM1 than neuronal cells, and catabolized it more extensively. After 14 h of incubation, non-neuronal cells retained only 14% of the TMR products as GM1 and GM2, compared to >50% for neurons. On average, non-neuronal cells contained 74% of TMR-labeled product as TMR-ceramide, compared to only 42% for neurons. Non-neuronal cells retained seven times as much TMR-GM3 (7%) compared to neuronal cells (1%). To confirm the observed single cell metabolomics, we lysed and compared TMR-GM1 catabolic profiles from mixed neuron/glial cell cultures and from cultures depleted of non-neuronal cells by treatment with the antimitotic agent cytosine arabinoside. The lysed culture catabolic profiles were consistent with the average profiles of single neurons and glia. We conclude that the ultrasensitive analytic methods described accurately reflect single cell ganglioside catabolism in different cell populations from the brain.     

Calcium: A novel and efficient inducer of differentiation of adipose‐derived stem cells into neuron‐like cells

This study comparatively investigated the effectiveness of calcium and other well‐known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose‐derived stem cells (ADSCs) into neuronal‐like cells. ADSCs were immunophenotyped and differentiated into neuron‐like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron‐specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real‐time polymerase chain reaction (quantitative real‐time polymerase chain reaction (qRT‐PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite‐like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron‐like cells and instantaneous increase in the expression of neuronal markers.     

A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow cytometry

High-throughput flow cytometry of adherent cells is difficult because the creation of single cell suspensions can damage cells and yield artificial results. We describe a protocol to increase the single cell suspension yield of adherent human cells without injury. Doxorubicin, a cytotoxic agent, was administered to adherent human pancreatic carcinoma cell lines (Panc-1 and AsPC-1) to produce alterations in the cell cycle and intracellular protein expression. The cells in 96-well plates were disassociated using a collagenase and trypsin mixture. Fluorescence-activated high-throughput flow cytometry evaluated cellular viability as well as surface and intracellular protein expression. Cell cycle analysis was performed using 7-aminoactinomycin D and intracellular protein characterization was performed using a fluorescein-labeled monoclonal antibody against activated caspase-3. The collagenase–trypsin-based protocol increased single cell events from 31.9 ± 0.5% using trypsin alone (standard) to a range of 62.1% to 85.5% without adversely affecting viability. High-throughput flow cytometry demonstrated that the addition of collagenase to the disassociation solution not only permitted significantly higher rates of single cell creation, but it did not negatively affect the doxorubicin-induced protein expression. This protocol allows for expedient and effective disassociation of adherent human cells in order to investigate alterations in specific cellular enzymes and pathways.     

Expression profiling of single cells using 3 prime end amplification (TPEA) PCR.

The ability to relate the physiological status of individual cells to the complement of genes they express is limited by current methodological approaches for performing these analyses. We report here the development of a robust and reproducible method for amplifying 3' sequences of mRNA derived from single cells and demonstrate that the amplified cDNA, derived from individual human lymphoblastoma cells, can be used for the expression profiling of up to 40 different genes per cell. In addition, we show that 3 prime end amplification (TPEA) PCR can be used to enable the detection of both high and low abundance mRNA species in samples harvested from live neurons in rat brain slices. This procedure will facilitate the study of complex tissue function at the cellular level.     

A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila

Mosaic analysis with a repressible cell marker (MARCM) is a genetic technique used in Drosophila to label single cells or multiple cells sharing a single progenitor. Labeled homozygous mutant cells can be generated in an otherwise unlabeled heterozygous animal. Mutant or wild-type labeled cells can also be made to express one or more transgenes. Major applications of MARCM include (i) lineage analysis, (ii) investigating gene function in single or small populations of cells and (iii) neuronal circuit tracing. Our laboratory uses MARCM primarily to label and genetically manipulate neurons; however, this protocol can be adapted to any cell of interest. The protocol involves generating two fly stocks with the necessary genetic elements for MARCM analysis and subsequently generating MARCM clones. Labeled clones can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells.NOTE: In the PDF version of this article initially published online, the first "FRT" and the "Mutation" labels in Figure 1b were transposed. In both the PDF and HTML versions, "mutant" was omitted from the label on the right, which should read "Labeled homozygous mutant daughter cell". The figure has been corrected in all versions of the article.     

Rapid isolation of single malaria parasite-infected red blood cells by cell sorting

Malaria research often requires isolation of individually infected red blood cells (RBCs) or of a homogenous parasite population derived from a single parasite (clone). Traditionally, isolation of individual, parasitized RBCs or parasite cloning is achieved by limiting dilution or micromanipulation. This protocol describes a method for more efficient cloning of the malaria parasite; the method uses a cell sorter to rapidly isolate Plasmodium falciparum-infected RBCs singly. By gating the parameters of forward-angle light scatter and side-angle light scatter in a cell sorter, singly infected RBCs can be isolated and automatically deposited into a 96-well culture plate within 1 min. Including a Percoll purification step; the entire procedure to seed a 96-well plate with singly infected RBCs can take <40 min. This highly efficient single-cell sorting protocol should be useful for cloning of both laboratory parasite populations from genetic manipulation experiments and clinical samples.     

Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line’s species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification.     

Methyl CpG Binding Domain Ultra‐Sequencing: a novel method for identifying inter‐individual and cell‐type‐specific variation in DNA methylation

Experience‐dependent changes in DNA methylation can exert profound effects on neuronal function and behaviour. A single learning event can induce a variety of DNA modifications within the neuronal genome, some of which may be common to all individuals experiencing the event, whereas others may occur in a subset of individuals. Variations in experience‐induced DNA methylation may subsequently confer increased vulnerability or resilience to the development of neuropsychiatric disorders. However, the detection of experience‐dependent changes in DNA methylation in the brain has been hindered by the interrogation of heterogeneous cell populations, regional differences in epigenetic states and the use of pooled tissue obtained from multiple individuals. Methyl CpG Binding Domain Ultra‐Sequencing (MBD Ultra‐Seq) overcomes current limitations on genome‐wide epigenetic profiling by incorporating fluorescence‐activated cell sorting and sample‐specific barcoding to examine cell‐type‐specific CpG methylation in discrete brain regions of individuals. We demonstrate the value of this method by characterizing differences in 5‐methylcytosine (5mC) in neurons and non‐neurons of the ventromedial prefrontal cortex of individual adult C57BL/6 mice, using as little as 50 ng of genomic DNA per sample. We find that the neuronal methylome is characterized by greater CpG methylation as well as the enrichment of 5mC within intergenic loci. In conclusion, MBD Ultra‐Seq is a robust method for detecting DNA methylation in neurons derived from discrete brain regions of individual animals. This protocol will facilitate the detection of experience‐dependent changes in DNA methylation in a variety of behavioural paradigms and help identify aberrant experience‐induced DNA methylation that may underlie risk and resiliency to neuropsychiatric disease.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

AHT-ChIP-seq: a completely automated robotic protocol for high-throughput chromatin immunoprecipitation

ChIP-seq is an established manually-performed method for identifying DNA-protein interactions genome-wide. Here, we describe a protocol for automated high-throughput (AHT) ChIP-seq. To demonstrate the quality of data obtained using AHT-ChIP-seq, we applied it to five proteins in mouse livers using a single 96-well plate, demonstrating an extremely high degree of qualitative and quantitative reproducibility among biological and technical replicates. We estimated the optimum and minimum recommended cell numbers required to perform AHT-ChIP-seq by running an additional plate using HepG2 and MCF7 cells. With this protocol, commercially available robotics can perform four hundred experiments in five days.