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Label Free Detection of Subtle Morphological Differences in Immune Cells

Sensitive and high-throughput technologies to assess subtle morphological differences in cells are a major need for high-throughput drug screening and disease profiling research. However, conventional technologies like flow cytometry and microscopy to assess morphology have limitations. Microscopy is slow and a terminal assay for cells while flow cytometry only provides crude physical measures, like size and granularity, by detecting forward (FSC) and side (SSC) scattered light, respectively. By these measures, subtle morphological differences between cells cannot be detected. However, the data-rich profiles generated by Ghost Cytometry enable detection and resolution of ultra-fine differences in cell morphology. In this proof-of-concept study we use the VisionSort platform, powered by Ghost Cytometry, to […]

Classification of Cellular Diversity and Disease Assessment in ALL

Acute Lymphoblastic Leukemia (ALL) is a malignancy of lymphoid progenitor cells in the bone marrow and blood. The disease has a bimodal distribution with 80% of ALL patients presenting as children who respond positively to therapy. However, for adult patients ALL can be a devastating disease, with only 30–40% achieving long–term remission. Chromosomal abnormalities, such as the Philadelphia chromosome, are commonly found in ALL and are a major determinant of prognostic outcome. While long-term survival in pediatric ALL without chromosomal abnormalities approaches 90%, Philadelphia chromosome positive adult ALL has a five-year survival rate of 5 – 20%. As research into the genomic determinants of ALL disease progression and response to […]

Label-free Macrophage Subtyping

As monocytes migrate into tissues, they differentiate into macrophages. Upon activation, macrophages can polarize and adopt two very different and biologically opposing phenotypes, termed M1 and M2. M1 macrophages are considered classically activated macrophages that mediate proinflammatory responses while M2 macrophages mediate anti-inflammatory responses, both by cytokine secretion. The balance of M1 and/or M2 macrophage polarization is known to be a mediator of physiological responses in tumor progression and as such, the ability to isolate cells in the two polarization states in purified populations is crucial for understanding the dynamics of tumor progression. We show here that VisionSort can be used to isolate pure populations of M1 and M2 macrophages […]

Regulatory T cell Characterization and Isolation

Regulatory T cells (Tregs) are specialized T cells that suppress immune responses by cytokine production and the inhibition of T cell proliferation. These specialized T cells are key for modulating the immune response and play a role in preventing autoimmunity. They also have a therapeutic role in preventing allograft rejection, allergies, and autoimmune diseases. Tregs have traditionally been defined by the expression of cell surface markers such as CD4 and CD25, and these labels are commonly used to identify these cells in flow cytometry applications.1 Cell therapy researchers need a supply of pure Treg cells, untouched by external labels, in order to perform functional studies and evaluate their utility as […]

T cell Activation

T cell activation is a critical step in the adaptive immune response. Studying the mechanisms of T cell activation is an important part of modern immunology research and in vitro activation is a requirement for cell therapy drug development programs. However, current methods to isolate activated T cells require labeling with T-cell markers (e.g. CD8) and key extracellular molecular activation indicators (such as CD38, CD45RO, and HLA-DR) with fluorescently tagged antibodies; a process that can interfere with downstream R&D assays and requires label removal in cell therapy development. The VisionSort platform enables label-free sorting of T cells, giving investigators and drug developers activated T cells, untouched by external labels, for […]

Label-Free High Throughput CRISPR-Based Screening for Morphological Phenotypes in Flow

The development of CRISPR-Cas9 genome editing technology has led to the emergence of a new generation of novel life sciences applications. In drug discovery, researchers have harnessed the precision of selective gene knockouts by CRISPR to enable genome-wide drug screening. By mapping genotypes to phenotypes, CRISPR-based phenotypic screens can enable a better understanding of drug mechanism of actions (MOAs) and identification of novel druggable targets. However, current phenotypic CRISPR screening approaches rely heavily on microscopic imaging of target phenotypes, a process that imposes throughput limitations and restricts screening to only a handful of simple phenotypes based on binary fluorescence signals. To fully realize the potential of CRISPRbased phenotypic screening, here […]

High Throughput CRISPR-Based Phenotypic Screening in Flow for Complex Intracellular Phenotypes

The development of CRISPR-Cas9 genome editing technology has led to the emergence of a new generation of novel life sciences applications1. In drug discovery, researchers have harnessed the precision of selective gene knockouts by CRISPR to enable genome-wide drug screening. By mapping genotypes to phenotypes, CRISPR-based phenotypic screens can enable a better understanding of drug mechanism of actions (MOAs) and identification of novel druggable targets. However, current phenotypic CRISPR screening approaches rely heavily on microscopic imaging of target phenotypes, a process that imposes throughput limitations and restricts screening to only a handful of simple phenotypes by binary fluorescence signals. To fully realize the potential of CRISPR-based phenotypic screening, here show […]

Intracellular Phenotyping with Enhanced Spatial Resolution in Flow

Many cellular processes and functions involve phenotypic changes or biomolecular interactions at the intracellular level. Researchers can exploit knowledge of these phenotypic changes to better elucidate basic cellular biology, profile disease pathology, or identify new drug targets. Classically, cellular phenotypes have been assessed using high-resolution, imagebased microscopy approaches. However, microscopy-based approaches are slow, manual, and time consuming. Recently, phenotypic assessment using flow cytometry has been developed, offering substantially higher throughput. However, conventional flow cytometers only collect data on the average intensity of light signals and can not deeply analyze intracellular phenotypes with a high degree of spatial resolution. In addition, imaging flow cytometers are unable to sort cells based on […]

Identification and Isolation of CAR-T Cells with Enhanced Therapeutic Efficacy

Chimeric antigen receptor T-cells (CAR-T) have emerged as an exciting new approach for the treatment of cancer, with over 500 CAR-T worldwide clinical trials underway in 2021.1 Conventional thinking was that all CAR-T cells have similar therapeutic potential, however new research has found that not all CAR-T cells are created equal. A recent study showed that CAR-T cells with lower levels of glycolysis led to better clinical responses in leukemia patients.2 Here we use VisionSort to identify metabolically distinct CAR-T cell subsets without the use of molecular labels or tags as an approach to identify cells with the potential for higher therapeutic efficacy.

Label-free Assessment of Cell Viability

Characterizing cell viability is critical for many life science research and development studies. Viability is typically assessed using fluorescent markers such as propidium iodine (PI) or 7-AAD, that exploit the loss of membrane integrity in dead cells which allows these markers to permeate into nuclei, bind double stranded DNA, and thus label cells in the late stages of death. In earlier stages of apoptosis where membrane integrity has not yet been lost, other fluorescent markers such as Annexin V, Apotracker, JC-1, Caspase-3/7 are commonly used to label activation of the apoptotic pathway. However, the use of fluorescent labels to identify cell viability states can hinder downstream research efforts and therefore […]

B cell Plasma Cell Differentiation

B cells are critical for a functional immune system. Activation of B cells results in a series of biochemical and cellular changes that convert naïve B cells to plasma cells. Differentiated plasma cells help the body to defend against foreign entities through the production of antibodies. This antigen-specific antibody production by plasma cells has also been exploited for the development of targeted therapeutics for cancer and other immune disorders. While B cells play a protective role in the adaptive immune system, dysfunctional B cells can lead to life threatening immunological disorders. Due to the importance of B cells for human health, studying the process of B-cell activation and plasma cell […]