Technological advances in fluorescence flow cytometry and an ever‐expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high‐quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high‐dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off‐peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high‐dimensional spectral flow cytometry panels using a comprehensive step‐by‐step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker‐fluorophore assignment is provided. © 2020 by John Wiley & Sons, Inc.

Technological advancements in fluorescence flow cytometry and an ever‐expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels reaching up to 43 colors at the single‐cell level. However, as panel size and complexity increase, so too does the detail involved in designing and optimizing successful high‐quality panels fit for downstream high‐dimensional data analysis. In contrast to conventional flow cytometers, full‐spectrum flow cytometers measure the entire emission spectrum of each fluorophore across all lasers. This allows for fluorophores with very similar emission maxima but unique overall spectral fingerprints to be used in conjunction, enabling relatively straightforward design of larger panels. Although a protocol for best practices in full‐spectrum flow cytometry panel design has been published, there is still a knowledge gap in going from the theoretically designed panel to the necessary steps required for panel optimization. Here, we aim to guide users through the theory of optimizing a high‐dimensional full‐spectrum flow cytometry panel for immunophenotyping using comprehensive step‐by‐step protocols. These protocols can also be used to troubleshoot panels when issues arise. A practical application of this approach is exemplified with a 24‐color panel designed for identification of conventional T‐cell subsets in human peripheral blood. © 2021 Malaghan Institute of Medical Research, Cytek Biosciences. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Preparation and evaluation of optimal spectral reference controlsSupport Protocol 1: Antibody titrationSupport Protocol 2: Changing instrument settingsBasic Protocol 2: Unmixing evaluation of fully stained sampleBasic Protocol 3: Evaluation of marker resolutionSupport Protocol 3: Managing heterogeneous autofluorescenceBasic Protocol 4: Assessment of data quality using expert gating and dimensionality reduction algorithms

Glioblastoma (GBM) causes poor survival in patients even with aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment but resistance always ensues. Protein kinase CK2 (CK2) contributes to tumour development and proliferation in cancer, and it is overexpressed in human GBM. Accordingly, targeting CK2 in GBM may benefit patients. Our goal has been to evaluate whether CK2 inhibitors (iCK2s) could increase survival in an immunocompetent preclinical GBM model. Cultured GL261 cells were treated with different iCK2s including CX-4945, and target effects evaluated in vitro. CX-4945 was found to decrease CK2 activity and Akt(S129) phosphorylation in GL261 cells. Longitudinal in vivo studies with CX-4945 alone or in combination with TMZ were performed in tumour-bearing mice. Increase in survival (p < 0.05) was found with combined CX-4945 and TMZ metronomic treatment (54.7 ± 11.9 days, n = 6) when compared to individual metronomic treatments (CX-4945: 24.5 ± 2.0 and TMZ: 38.7 ± 2.7, n = 6) and controls (22.5 ± 1.2, n = 6). Despite this, CX-4945 did not improve mice outcome when administered on every/alternate days, either alone or in combination with 3-cycle TMZ. The highest survival rate was obtained with the metronomic combined TMZ+CX-4945 every 6 days, pointing to the participation of the immune system or other ancillary mechanism in therapy response.