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Many laboratories struggle with mycoplasma contamination and cell line misidentification when growing cells in culture. These well-documented issues affect the scientific research community and have detrimental downstream effects. Research published with suspect cultures can produce misleading results. There is increasing pressure to verify the integrity of experimental and established cell lines before publishing. Therefore, laboratories need to define how and when to perform these critical tests, analyze the results, and determine action plans if disparities exist. Our laboratory is committed to producing cell lines of the highest quality for use in experiments; thus, we created a surveillance strategy for these potential problems. We developed processes for both testing and tracing cell line authentication and mycoplasma detection data. Using these methods, we can protect the integrity of our patient and commercial cell lines, maintaining reliable cultures for our research.

Background: The ability of 3D printing using plastics and resins that are magnetic resonance imaging (MRI) compatible provides opportunities to tailor design features to specific imaging needs. In this study an MRI compatible cradle was designed to fit the need for repeatable serial images of mice within a mouse specific low field MRI., Methods: Several designs were reviewed which resulted in an open style stereotaxic cradle to fit within specific bore tolerances and allow maximum flexibility with interchangeable radiofrequency (RF) coils. CAD drawings were generated, cradle was printed and tested with phantom material and animals. Images were analyzed for quality and optimized using the new cradle. Testing with multiple phantoms was done to affirm that material choice did not create unwanted image artifact and to optimize imaging parameters. Once phantom testing was satisfied, mouse imaging began., Results: The 3D printed cradle fit instrument tolerances, accommodated multiple coil configurations and physiological monitoring equipment, and allowed for improved image quality and reproducibility while also reducing overall imaging time and animal safety., Conclusions: The generation of a 3D printed stereotaxic cradle was a low-cost option which functioned well for our laboratory., (© 2021. The Author(s).)

Sharing physical laboratory space optimizes resource use among research departments, improves collaboration, and reduces costs. Universal inventory management is essential to successful space sharing, item tracking and availability, and data mining; therefore, our lab was interested in a tool for these purposes. Different research groups have differing needs, priorities, and considerations. We investigated prebuilt inventory management programs, and we found a custom Microsoft (MS) Access reference database to be optimal. We designed a custom MS Access abstract inventory data model around previously established needs with information gathered in staff interviews and meetings. We performed a physical inventory of all cryopreserved samples to allow uniform data entry when migrating to the database. We created manuals and protocols, and the staff was trained on entering data. Using a universal inventory system allows for quick report generation of physical inventory, which in turn preserves sample quality by reducing redundancy as well as time locating vials. Users can run queries to report a wide range of sample characteristics. Storage information can be easily analyzed for statistically significant trends, samples have reliable traceability, and data are auditable for accuracy.

Background: CTLA-4 was initially described as a membrane-bound molecule that inhibited lymphocyte activation by interacting with B7.1 and B7.2 molecules on antigen presenting cells. Alternative splicing of mRNA encoding the CTLA-4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. Despite studies finding that people with autoimmune disease more frequently express high levels of sCTLA-4 in their blood than apparently healthy people, the significance of these findings is unclear., Methods: Molecules isolated from blood using CTLA-4 specific antibodies were analyzed with ligand binding assays, mass spectroscopy, and biochemical fractionation in an effort to increase our understanding of CTLA-4 immunoreactive material., Results: Mass spectroscopy analysis of the molecules recognized by multiple CTLA-4-specific antibodies failed to identify any CTLA-4 protein. Even though these molecules bind to the CTLA-4 receptors B7.1 and B7.2, they also exhibit properties common to immunoglobulins., Conclusion: We have identified molecules in blood that are recognized by CTLA-4 specific antibodies but also exhibit properties of immunoglobulins. Our data indicates that what has been called sCTLA-4 is not a direct product of the CTLA-4 gene, and that the CTLA-4 protein is not part of this molecule. These results may explain why the relationship of sCTLA-4 to immune system activity has been difficult to elucidate.

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4-Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4-Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.