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We have developed a semester-long laboratory project for an undergraduate immunology course in which students study multiple aspects of macrophage biology including differentiation from progenitors in the bone marrow, activation upon stimulation with microbial ligands, expression of cell surface markers, and modulation of cytokine production. In the first part of the semester, students differentiate macrophages from mouse bone marrow stem cells and perform immunophenotyping on their macrophages using myeloid markers that are either constitutively expressed or expressed upon activation with microbial ligands. Students use a low-cost image cytometer to both visualize and quantify cellular expression of myeloid markers. Students then perform literature research, design, and execute a series of experiments aimed at investigating the role of natural anti-inflammatory compounds on TNF- production in these macrophages. The soup-to-nuts investigative approach in which students generate "their own" macrophages and study their functions over the course of the semester fostered a sense of ownership and accomplishment

Immunosuppressive cells accumulating in the tumor microenvironment constitute a formidable barrier that interferes with current immunotherapeutic approaches. A unifying feature of these tumor-associated immune and vascular endothelial cells appears to be the elevated expression of ectonucleotidase CD39, which in tandem with ecto-5'-nucleotidase CD73, catalyzes the conversion of extracellular ATP into adenosine. We glycoengineered an afucosylated anti-CD39 IgG2c and tested this reagent in mouse melanoma and colorectal tumor models. We identified major biological effects of this approach on cancer growth, associated with depletion of immunosuppressive cells, mediated through enhanced Fcy receptor-directed (Fc[gamma]Rdirected), antibody-dependent cellular cytotoxicity (ADCC). Furthermore, regulatory/exhausted T cells lost CD39 expression, as a consequence of antibody-mediated trogocytosis. Most strikingly, tumor-associated macrophages and endothelial cells with high CD39 expression were effectively depleted following antibody treatment, thereby blocking angiogenesis. Tumor site-specific cellular modulation and lack of angiogenesis synergized with chemotherapy and anti-PD-L1 immunotherapy in experimental tumor models. We conclude that depleting suppressive cells and targeting tumor vasculature, through administration of afucosylated anti-CD39 antibody and the activation of ADCC, comprises an improved, purinergic system modulating strategy for cancer therapy., Introduction Cancer treatment has been radically transformed by immunotherapies that can unleash antitumor immune responses, as highlighted by the utility of checkpoint blockade antibodies against PD-1/ PD-L1 and CTLA-4. These [...]

Mixed microorganism cultures are prevalent in the food industry. A variety of microbiological mixtures have been used in these unique fermenting processes to create distinctive flavor profiles and potential health benefits. Mixed cultures are typically not well characterized, which may be due to the lack of simple measurement tools. Image-based cytometry systems have been employed to automatically count bacteria or yeast cells. In this work, we aim to develop a novel image cytometry method to distinguish and enumerate mixed cultures of yeast and bacteria in beer products. Cellometer X2 from Nexcelom was used to count of Lactobacillus plantarum and Saccharomyces cerevisiae in mixed cultures using fluorescent dyes and size exclusion image analysis algorithm. Three experiments were performed for validation. (1) Yeast and bacteria monoculture titration, (2) mixed culture with various ratios, and (3) monitoring a Berliner Weisse mixed culture fermentation. All experiments were validated by comparing to manual counting of yeast and bacteria colony formation. They were highly comparable with ANOVA analysis showing p-value > 0.05. Overall, the novel image cytometry method was able to distinguish and count mixed cultures consistently and accurately, which may provide better characterization of mixed culture brewing applications and produce higher quality products. [ABSTRACT FROM AUTHOR]

In the recent decade, chimeric antigen receptor (CAR)‐T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. The success of CAR‐T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Image cytometry methodologies have been utilized for various CAR‐T cell‐mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease‐of‐use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo Image Cytometer to evaluate and compare two CAR‐T cell‐mediated cytotoxicity assays using GFP‐expressing or fluorescent dye‐labeled myeloma and plasmacytoma cells. The GFP‐based method demonstrated higher sensitivity in detecting CAR‐T cell‐mediated cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit‐for‐purpose to ensure the results produced are meaningful for the specific testing conditions. [ABSTRACT FROM AUTHOR]