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CAR-MA Assays

All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

As a world-renowned service provider for immunotherapy, Creative Biolabs has successfully established genetic engineering chimeric antigen receptor macrophage (CAR-MA), aiming to improve the ability of CAR cells to attack solid tumors. Here, we have updated a comprehensive set of CAR-MA functional assays to meet our clients' academic or clinical purposes.

CAR-MA is an innovative and exciting therapeutic technology that uses unexpanded fresh T cells and other mononuclear immune cells to respond to cancer cells. Animal studies have shown that producing a large number of targeted immune cells and delivering them to cancer cells effectively kills targeted cancer cells with high specificity and appears to be a safe and effective cell therapy for solid tumors. Therefore, further development of CAR-MA could be useful on several therapeutic fronts, which has the potential to provide cell therapies with fewer adverse events than traditional cell therapies.

Anti-tumour response propagated by CAR-MAs within the tumor environment. Fig.1 Anti-tumour response propagated by CAR-MAs within the tumor environment. (Qin, 2021)

We offer CAR-MA-based assays to support customers' flexible choices.

  • CAR expression detection
  • The expression of CAR on the cell surface is a key feature of active CAR-MA products. Evaluating CAR expression is an essential step in the production of CAR cells. We provide accurate and reproducible CAR detection methods to support the development of CAR-MA therapy.

  • Bead engulfment assay - CAR expression validation
  • To assess the engulfment of macrophages expressing the appropriate CAR, we use silica beads of diverse diameter coated with a supported lipid bilayer as the engulfment target. To further validate the ability of CARs to redirect primary macrophages, our engineered primary macrophages are ready for CAR-MA expression, and we will evaluate the engulfment efficacy of the transfected primary cells.

  • Bites assay - CAR-MA specificity and function measurement
  • To assess the antigen specificity of CAR-MA, we incubate CAR-MA in our extensive antigen pool to detect which antigens CAR-MA is most likely to bite. It's worth noting that our expertise enables the design of custom antigens to meet your specific research needs. To further define the capabilities of CAR-MA, an assay for the capacity of CAR-MAs to bite variably sized target beads will be performed. Creative Biolabs possess highly specific beads above background engulfment ranging from 2.5 mm to 20 mm in diameter.

  • Eating assay - Phagocytosis test against the target cell
  • To assess CAR-MA engulfment towards a cellular target, CRAMA internalized eating of the target cell will be tested by incubating the CAR-MA with target cells that express high levels of endogenous antigens. To enhance whole-cell eating, our talented technicians will design specific strategies to improve the efficiency of whole eating, from chimeric antigen receptors to antibodies.

  • Confocal imaging
  • All images in the CAR-MA assay will be performed using an environmentally controlled rotating disk confocal microscope (Nikon Ti-Eclipse inverted microscope with Yokogawa rotary disk unit). For bead internalization measurements, we use 40×0.95 N/A air objective to capture images and use the High Content Screening (HCS) Site Builder for unbiased real-time image acquisition. In addition, we also use the 100×1.49 N/A oil immersion objective to obtain additional images. All images will be acquired using an AndoriXon EM-CCD camera.

  • Quantification of whole-cell internalization
  • We inoculate macrophages into glass-bottom MatriPlate. Before imaging, LPS will be used to stimulate the macrophages and then quantify their beads' engulfment. Delay analysis is essential to ensure that beads appear to be viable prior to engulfment by the macrophage.

  • Quantification of bites internalization
  • During live-cell image acquisition, GFP-positive macrophages will be selected by the presence of GFP signal. A full image comprising the entire cell will be captured using 1 mm steps and then manually inspected for internalized beads. Cells containing one or more bites of fully internalized beads>1 mm in diameter are scored as positive.

  • FRET assay
  • FRET has become a powerful tool for detecting nanometer-scale distance changes of biological macromolecules in living organisms and has been widely used in cell physiology research and immunoassay. Our FRET assay allows you to detect the interactions between the molecules you are interested in.

  • CAR-MA cytokine release assay
  • Macrophages release cytokines via a series of beautifully orchestrated pathways that are spatiotemporally regulated. Our platform provides standard methods to measure the effector cytokines responses, such as enzyme-linked immunospot assay (ELISpot) and intracellular cytokine staining assay (ICS).

  • CAR-MA cell phenotype analysis
  • Macrophages respond in a highly plastic manner to external perturbations and consequently differentiate into phenotypically distinct subpopulations. The polarisation of the macrophage is commonly classified into two distinct states: the classically activated M1 macrophage and the alternative activated M2 macrophage. There are many markers identified to analyze the phenotypes of macrophages. We provide services to assess phenotypic markers by flow cytometry, ELISA, and qPCR.

  • Antigen cross-presentation assay
  • To test CAR-MA cross-presentation of intracellular tumor antigens ingested during phagocytosis of whole tumor cells, we incubate T cell with CAR-MA treated with tumor antigen. And then we test the potential of CAR-MA to stimulate T cells by T cell proliferation assay and cytokines release assay.

  • CAR-MA immunometabolism analysis
  • Macrophages shift their metabolism in response to environmental cues. In turn, these metabolic adaptations drive specific effector functions in macrophages. At Creative Biolabs, we provide multiple immunometabolism analyses including metabolomics services and various metabolism assay kits.

CAR-MA services and related assays are Creative Biolabs' CAR-based cell therapy proprietary services. Our experienced scientists will greatly facilitate you to achieve highly unmet needs, particularly in cell therapy. Please contact us for more information and a detailed quote.

Reference

  1. Qin, V.M.; et al. Chimeric antigen receptor beyond CAR-T Cells. Cancers. 2021, 13(3): 404.
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