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The use of conventional serum for supplementation of media in cell-based and single-cell functional assays has been a major challenge for assay performance, standardization, optimization, and reproducibility. It has been identified as the leading cause of variability and suboptimal performance in large, international Elispot proficiency panels (Janetzki et al., 2008; Rountree et al., 2016). Extensive pretesting and optimization activities are one approach to overcome these challenges, but they are time-consuming and resource-intensive because suitable lots of serum are difficult to identify and secure in sufficient quantities to provide stability in long-term studies. Advancements in manufacturing methods have resulted in a new class of serum with the potential to solve these challenges. An IFNɣ Elispot study was designed by the External Quality Assurance Program Oversight Laboratory (EQAPOL) at Duke Human Vaccine Institute's (DHVI) Immunology and Virology Quality Assessment Center (IVQAC) to test this new class of serum against their in-house, validated control serum, which is regarded as a global standard in performance for high functionality, recovery, and viability. Commonly used serum-free media were also included in the study. The results of this study compellingly demonstrate that this new class of serum produces high responses and low background reactivity comparable to the included serum standard, with excellent recovery and viability of cells. A protocol for ongoing testing has been developed to continuously validate new batches of this serum with the goal to make available to the field a pretested and validated serum that can be used with confidence in functional cell-based assays., Competing Interests: Declaration of Competing Interest Guido Ferrari reports financial support and equipment, drugs, or supplies were provided by Seraworx, LLC. Devin Davis reports a relationship with Seraworx, LLC that includes: employment and equity or stocks. Sylvia Janetzki reports a relationship with Seraworx, LLC that includes: consulting or advisory., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The ELISpot assay prevails as one of the most sensitive and meaningful assays for the detection of antigen-specific, effector immune responses. Acquisition of cellular analyte for ELISpot analysis is typically not problematic when derived from tissues enriched in lymphocytes (e.g., lymphoid organs and blood); however, cell processing becomes more difficult when lymphocytes represent only a very minor population relative to the source tissue, especially when the source tissue is in limited supply (e.g., small mouse tumors). Traditional enzymatic-based methods for dissociating tumors often result in poor yields, inconsistent lymphocyte enrichment, and can have deleterious effects on lymphocyte phenotype and function. To address these limitations, we have developed an enzyme-free protocol for processing tumor infiltrating lymphocytes (TILs) from small mouse tumors, which enables the enumeration of antigen-specific effector lymphocytes using ELISpot analysis. This procedure is predicated on the dissociation of tumor tissue using gentle agitation with a paddle blender followed by a brief in vitro culture period to remove adherent cells, as well as to revive lymphocytes from a non-responsive state. Although this method is demonstrated with mouse intracerebral tumors, we have found that this protocol is applicable to peripheral tumors and may likely extend to alternative tissue sources wherein lymphocytes exist in low numbers., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)