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The single cell resolution is paving the way towards the molecular characterizations of every cell type in different contexts. The application of single cell genomics technologies has revolutionized the existing approach to explore an individual's immunity. Unfolding the functional diversity of immune cells and their coordinated reaction is key to understanding the immune system. Single cell transcriptomics technologies open up a high-dimensional assessment of the transcriptional state of immune cells and have been successfully used to discover novel immune cell types, reveal hematopoietic lineages, identify gene modules regulating immune responses, and analysis lymphocyte antigen receptor diversity.

As a famous and reliable partner in the world, SingleX pays close attention to the impact and applications of single cell multi-omics and RNA-sequencing technologies in immunology. We're skilled at conducting real-time monitoring of immunity on the level of individual cells, and the advent of our powerful single cell assays has allowed clients to exploit developmental trajectories during immune responses.

Monitoring of Immunity

Tracing Immune Lineage through Clonality

In humans, a cell acquires some innocuous mutations per cell division, making mutation-based lineage reconstruction at the single cell level possible. Genome and transcriptome sequencing (G&T-seq) can measure the genomic and transcriptomic sequences of single cells at the same time, with a coverage of less than 70%.

Also, immune cell lineages can be inferred by introducing cell-to-cell genomic variability, using a suite of recently developed ways, genetic scarring. The methods insert mutations in a defined sequence to produce a synthetic barcode or a transgene. Most of the approaches utilize CRISPR/Cas9 to generate the mutation in vitro. An alternate approach combines Cre-loxP recombination with an artificial DNA recombination locus (Polylox) to enable genetic barcoding.

For T cells, several algorithms have been designed to infer the T-cell receptor (TCR) sequence from single cell RNA-sequencing data and then reconstructing a clonal network. For B cells, the process of somatic hypermutation and isotype switching further complicate the reconstruction of B-cell receptor (BCR) sequences, requiring additional computational protocols and bioinformatic analysis. Algorithms for this have been developed and will be helpful in the development and immune response of B cells.

Dissecting Molecular Pathways of Immune Responses

The study of cellular trajectories and molecular pathways has a deep history in the field of immunology. It is largely due to the ease with which freely mobile immune cells can be analyzed by fluorescence-activated cell sorting (FACS). This method uses fluorochrome-labeled antibodies against surface or cellular molecules, together with other fluorescent proteins and dyes, to evaluate cellular phenotypes. FACS has been performed to study the activation of immune cells to stimulus and is particularly useful in revealing the correlation between cell division and differentiation. Theoretically, this instrument allows for simultaneous assessment of 50 parameters.

Time-lapse microscopy excels at the analysis of a few cells in vitro and permits the continual visualization of immune cells in real time. Unlike other techniques, it can be used as direct evidence of transitioning cells, and provide temporal information about these current changes. Remarkably, intravital two-photon microscopy overcomes some limitations of standard microscopy by improving the imaging of cells in situ and has been implemented to dissect their association with lymphocytes in response to immune challenge. Besides, the emerging of next-generation sequencing (NGS) to measure transcriptome at single cell level has fascinated our ability to interrogate the immune system.

Monitoring of Immunity
Monitoring of Immunity

Unravelling Antigen Receptor Diversity at Single Cell Level

TCR is the mediator of specific antigen recognition by T cells, that is formed by the random rearrangement of genomic V(D)J (variable(diversity)joining) segments. This process is directly analogous to the generation of antibody diversity by somatic V(D)J recombination of the BCR locus in B cells. The TCR compose of a heterodimer of two chains (αβ or γδ), both of which are products of V(D)J recombination. The diversity of TCR repertoires, as generated by somatic DNA rearrangements (only in), is critical to immune system function. It occurs only in the T-cell genome and can be effectively used as a unique tag to enumerate and quantify.

Using techniques such as PCR, Southern blot, and flow cytometry, we have characterized T-cell or B-cell proliferation. Today, high throughput sequencing technology offers significant advantages over these approaches and has enabled examination of antigen receptor repertoires at single-nucleotide and, more recently, at single-cell resolution. This measurement provides extensive, comprehensive analyses of the diversity of both TCR and BCR to elucidate immune functions in health and disease.

Highlights

  • An end-to-end solution, cost-effective plan, and reduced timeline.
  • Multiple strategies to satisfy varying objectives for immunity monitoring.
  • Full deliverables with a detailed report and bioinformatic analysis.

The immunity is under constant pressure to defend the body against internal and environmental threats. In the era of precision medicine, single cell multi-omics methods enable the integration of data from diverse omics platforms, bringing multi-insight into the interrelation of these omics layers on immune processes. With expertise in system biology and rich experience in single cell assays, SingleX focuses on single cell omics experimental and computational approaches for unraveling cellular lineage and antigen receptor diversity in the context of immunology. Please don't hesitate to contact us to know more.

! ! For research purposes only. Not for clinical, therapeutic, or diagnostic purposes in animals or humans.

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