Humidified CO 2 incubator. Critical notes Resuspend the Dynabeads in the vial carefully before use, i. This product should not be used with Dynal MPC Never use less than the recommended volume of Dynabeads.
Carefully follow the recommended pipetting volumes. Avoid air bubbles during pipetting. Prior to flow cytometric analysis, Dynabeads and bead-bound cells should be removed. Upon activation and for days thereafter, some cells will bind strongly to the beads. The bead-bound cell fraction can be cultured overnight and the above process repeated to further increase T cell recovery.
When using cells for proteomics or gene expression studies, lyse the cells prior to bead removal. Preparations See www. Prepare cell culture medium Dynabeads Washing Procedure Dynabeads should be washed before use. Transfer the desired volume of Dynabeads to a tube.
Add an equal volume of Buffer, or at least 1 ml, and mix vortex for 5 seconds, or keep on a roller for at least 5 min. Place the tube on a magnet for 1 min and discard the supernatant. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of Culture Medium as the initial volume of Dynabeads taken from the vial step 2.
Harvest the activated T cells and use directly for further analysis. For flow cytometry applications, remove the beads prior to staining. Place the tube on a magnet for minutes to separate the beads from the solution. Expansion of Human T Cells Start with Examine cultures daily, noting cell size and shape.
Cell shrinking and reduced proliferation rate is typically observed in exhausted cell cultures. Count the cells at least twice weekly after thorough re-suspension. When the cell density exceeds 2. Certification Invitrogen Dynal AS conforms to the Quality Systems Standard ISO and ISO with the following scope: "Development, manufacturing, marketing and sales of Dynospheres, Dynabeads and associated products to customers that work within immunology, biological and clinical research, cell based therapy and in vitro diagnostics.
Volpe E et al. De Fanis U et al. Blood 10 Schade AE et al. Product Applications This product is designed for use in the following research area s as part of the highlighted workflow stage s. Research Area. Workflow Stages for T Cell Research. Cell Sourcing. Cell Isolation. Cell Activation, Expansion, and Differentiation. Cell Characterization. Workflow Stages for T Cell Engineering.
Cell Cryopreservation. View All. Data and Publications Data. Gao et al. View publication. Abstract Transcriptional status determines the HIV replicative state in infected patients. However, the transcriptional mechanisms for proviral replication control remain unclear.
Targeting these mechanisms may have a therapeutic potential to eradicate HIV infection. Trapecar et al. Abstract Although the association between the microbiome and IBD and liver diseases is known, the cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver, and circulating Treg and Th17 cells, we created a multi-organ model of ulcerative colitis UC ex vivo.
Using multiomics, we found SCFAs increased production of ketone bodies, glycolysis, and lipogenesis, while markedly reducing innate immune activation of the UC gut. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism, and tissue homeostasis.
Schwinn et al. Abstract The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology.
For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays.
In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches. Cerezo et al. Abstract Preventing the immune escape of tumor cells by blocking inhibitory checkpoints, such as the interaction between programmed death ligand-1 PD-L1 and programmed death-1 PD-1 receptor, is a powerful anticancer approach.
However, many patients do not respond to checkpoint blockade. Tumor PD-L1 expression is a potential efficacy biomarker, but the complex mechanisms underlying its regulation are not completely understood. Thus, eIF4A inhibitors, in development as anticancer drugs, may also act as cancer immunotherapies.
Li et al. Abstract Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression.
In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.
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