Title: The effect of reference control material on producing usable data.

Author: Carissa Moore, Ph.D. Flow Contract Site Laboratory, LLC

Abstract: Advances in spectral flow cytometry have led to a revolution in how much information can be generated from a single sample. However, as the panels increase in complexity, the challenges to generate interpretable data also increase. Part of the challenge in spectral flow cytometry is in understanding how to create controls and what was standard practice for traditional flow cytometry is not acceptable for spectral flow cytometry. To understand how reference controls can affect unmixing of a sample and the results generated, three different materials were used to generate reference controls: Whole blood, compensation beads, and cell-like Slingshot beads. To make sure that we could see obvious unmixing errors, a purposely “bad” 15- color panel was designed that broke several rules for spectral panel design. All three sets of materials were stained with single colors to create the three sets reference controls. The three sets of reference controls and a full stained whole blood sample were run on a 4-laser Cytek Aurora and then unmixed the same sample with each set of reference controls. The resulting data was evaluated by NxN plots to look for unmixing errors that could cause inaccurate data or excessive manual compensation. T cell, NK cell, and B cell lymphocyte populations were gated to show the data that each unmixing would produce. The data show that traditional compensation beads caused large unmixing errors that would require extensive manual compensation adjustments and led to unusable data. The Slingshot beads caused fewer unmixing errors, but the cell-based reference controls gave the fewest errors. However, even with cell-based reference controls, unmixing errors that would require minor manual compensation are present due to the poor panel design. When branching into spectral flow cytometry, it is important to understand the contribution of quality reference controls to the success of your experiment. At FCSL, cellular based reference controls are used primarily with Slingshot beads for very rare markers that are not present in normal samples. Traditional compensation beads are no longer used to create reference controls as they cause too many unmixing errors.

Title: GMP-compliant cell sorting and expansion of CD137+ Tumor Infiltrating Lymphocytes (TILs) with specific reactivity against autologous tumor cells

Author: Daniela Andrade, Miltenyi Biotec

Abstract: Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has been shown to be a promising strategy for the treatment of advanced metastatic melanoma. This potent therapeutic approach requires easy and effective isolation of TILs in compliance with GMP cell manufacturing requirements. Here, we demonstrate the generation of tumor-reactive CD137+ lymphocytes with high purity and specific reactivity against autologous tumor cells upon further expansion. As a starting material, metastatic melanoma samples were dissociated, and tumor-infiltrating T cells were outgrown for two weeks in a high-dose IL-2 culture. Cells were frozen for shipping. Thawed outgrown TILs were stimulated overnight with autologous tumor cells. In order to enrich tumor-reactive T cells within the sample, cells were stained with GMP CD4-VioBlue, GMP CD8-APC, GMP CD137-PE and sorted using MACS® GMP Tyto® Cartridge, MACS® GMP Tyto® Running Buffer and MACSQuant® Tyto® Cell Sorter. The sorted tumor-reactive CD137+ T cells, at a purity of 96%, were transferred to the CliniMACS Prodigy® TRT System. Cell numbers increased from 7×105 CD137+ TRTs to 1.72×109 cells within 14 days of T cell culture with the CliniMACS Prodigy® TRT System, reflecting a 2457-fold expansion. As a control, nonsorted TRTs were expanded using the same methodology, showing an increase from 1×106 cells to 1.46×109 cells over the 14-day expansion period (1460x-fold). At the end of the culture, the frequency of CD8+ among CD3+ T cells was 99% among the Tyto-sorted TRTs and 93% in the non-sorted TRTs, showing a robust expansion and of CD8+ T cells and high viability (98%) in both cultures. While at the beginning of the expansion, the effector memory population was dominating, the central memory fraction increased throughout the expansion and comprised 50% of Tyto-sorted and 41% of non-sorted TRTs at day 14. In line with this observation, expression of exhaustion markers TIM3, LAG3, and PD1+ was down-regulated on day 14 compared to day 0 and day 7 and was similar between sorted and non-sorted cells. After 14 days of expansion, the Tyto-sorted TRTs showed a higher activation (43% for INFγ + and 35% for CD107a+) against the autologous tumor cells than the non-sorted TRTs (12% INFγ+, 12% CD107a+). A higher frequency was also observed for the activation marker CD137 (57% for the Tyto-sorted TRTs vs. 29% for the non-sorted TRTs). Overall, a robust reactivity against the autologous tumor cells with minimal alloreactivity could be achieved. In conclusion, our results present a new workflow for the isolation of tumor-reactive T cells from metastatic melanoma samples with high purity, and robust expansion of highly purified tumor-reactive T cells with specific reactivity against autologous tumor cells.

Title: Quality and Internal Control of Two Spectral Flow Cytometry Panels Across Four Months

Author: Amber Leonard, Benaroya Research Institute

Abstract: In an ex vivo study using deep immunophenotyping of human peripheral blood mononuclear cells (PBMCs), samples were acquired over a four-month period. To mitigate variability in the flow cytometer on each acquisition day, 8-peak beads were used to adjust all forty-eight parameter’s voltages, so the geometric mean fluorescence intensity (MFI) matched the 8-peak bead MFIs taken the same day as the reference controls were acquired. Reference controls were acquired once upon commencement of the study. Those same reference controls were used in spectral unmixing for each subsequent batch of samples acquired over the four months. In addition, for each batch of PBMCs acquired, an internal control (a single person’s PBMCs) was assayed and acquired alongside the participant samples. This allowed for continuous assessment of staining variability and machine performance.

Title: Viability Dye Stability for use in Flow Cytometry Applications

Author: Sarah Cocris, Flow Contract Site Laboratory, LLC

Abstract: Background: In the field of Flow Cytometry, the ability to distinguish live cells from dead or dying cells is essential for accurate analysis. This differentiation is commonly achieved through viability staining, utilizing amine reactive dyes. These dyes typically come with a manufacturer’s expiration of 2 weeks to 1 month following reconstitution. However, in scenarios involving low-frequency testing, adhering to these shelf lives is not economically advantageous. This research aims to extend the expiration dates in our lab past the manufacturer’s recommendations. Method: Aliquots of PBMC’s in a ratio of 30:70 (heat-killed to live cells) were frozen and stored at -80°C. Lyophilized vials of Zombie NIR, Zombie Yellow, Zombie Green, Live/Dead NIR, and Live/Dead Aqua were resuspended in 100µL of pre-warmed DMSO and then aliquoted and frozen at -80°C. This study consists of 8 timepoints, with the potential for extension up to 12, allowing for a comprehensive data set over the course of a full year. At each time point, 1 vial of PBMC’s and 1 vial of each dye were thawed. PBMC’s were washed and split into 7 tubes at 100µL each (1 unstained, 5 with corresponding dyes 1µL each, and 1 with 2.5µL of unexpired 7-AAD). Live/dead staining was performed with relative percent and mean fluorescence intensity evaluated using the BD FACSCanto to observe differences, if any, over time. Results: Over time, there is a decrease in mean fluorescence intensity (MFI); however, the differentiation between live and dead cells remains evident on cytograms. Conclusion: Expiration of amine reactive dyes can be extended past manufacturer recommendations.

Title: The Accellix Platform: Small and simple – an automated flow cytometer and a single use cartridge for sample preparation

Author: Mark Rehse, Accellix

Abstract: The Accellix Platform: Small and simple – an automated flow cytometer and a single use cartridge for sample preparation. The successful development and commercialization of cell therapies has provided hope for patients with urgent medical needs. The manufacturing of cell therapies is very complex, and their quality must be controlled by performing a set of tests to ensure identity and potency. Traditional flow cytometry is the gold standard for cell phenotyping, enumeration, and characterization. And incorporating the method into a commercially viable cell therapy manufacturing workflow is challenging, due to the complexity and time-intensive nature of the operation and maintenance of traditional flow cytometers, and the growing complexity of data analysis The Accellix Platform is a measurement and analysis system composed of a compact benchtop flow cytometer instrument and a single-use cartridge. Here we discuss the Accellix, a single 488 nm blue laser spectral flow cytometer with a silicon photomultiplier detector capable of the detection of 11 channels. The Accellix does not require any manual pulse amplification or compensation, so the platform operation is easy and requires minimal training. One of the main challenges facing the successful development and commercialization of drug therapies is the high degree of variability and irreproducibility that traditional flow cytometry cannot resolve easily, this is due in part to, regents; lot-lot variability, human error; pipetting, analysis and reporting, and non-optimized protocols. Accellix has strived to offer a complete system to help reducing these variabilities. At Accellix we offer • Dried down reagents in a tube format that have undergone rigorous testing to ensure consistency and stability. Dried down antibodies, control beads and all solutions required for the flow assay are integrated into a single-use, QR coded customized cartridge. The cells flow through the microfluidic channels of the cartridge that includes a 50 µM reading cuvette where cells are illuminated one by one by the laser. • We offer several OTS panels for routine applications such as TBNK, T-cell, Stem Cells and Custom Cartridges to customers built to their specifications by incorporating their specific antibodies into the panels. • Lastly, Accellix offers customized automated data analysis (auto classification) this means that there is no need for the operator to manually analyze the data. Accellix’s fully automated data analysis and auto classification process does not require manual data analysis.

Title: Classification of Imaging Flow Cytometry Micronucleus Data Using the Convolutional Neural Network and Random Forest Algorithms

Author: Raymond Kong, Cytek Biosciences

Abstract: The in vitro micronucleus (MNi) assay is a well-established quantitative assessment for cellular DNA damage and is essential to numerous fields, including genetic toxicology during drug development and bio-dosimetry during radiation exposure. The MNi assay is typically scored by manual microscopy, in which the results lack accuracy and precision due to scorer variability and fatigue. Alternatively, methods such as automated slide-scanning microscopy, which lacks cytoplasmic visualization, and conventional flow cytometry, which does not provide visual confirmation of MNi, may not be desirable. Recently, a rapid and automated MNi assay based on high-throughput image capture and feature-based image analysis using the Cytek® Amnis® ImageStream®X Mk II (ISX) imaging flow cytometer and IDEAS® software has been developed and optimized to overcome these limitations. However, this IDEAS® feature-based approach requires expertise in designing custom masks and features. As a result, a deep learning method based on convolutional neural networks (CNN) using Amnis® AI 1.0 software was validated to score imaging flow cytometry MNi assay data in a study using several chemicals tested across multiple cell lines (reported in CYTO 2022). Amnis® AI 2.0 now includes the Random Forest (RF) algorithm. While the CNN handles all spatial information of the input images without additional user intervention, such as new feature creation, the RF discerns object relationships among existing numeric features with the use of decision trees. Here, we showcase the CNN and RF algorithms in training artificial intelligence models to analyze ISX imagery of L5178Y cells treated with Mitomycin C (MMC). The CNN and RF performances are compared in terms of class prediction probabilities in classification experiments. In addition, the robustness of the feature-based RF is further evaluated by comparing the results obtained from a RF model using all the IDEAS ® default and custom features for the assay versus the RF results generated by a RF model with the top performing or custom features removed from the data files. These findings provide insights for Amnis® AI users in choosing the most appropriate algorithm for classifying imaging flow cytometry data in the MNi assay or other applications.

Title: Optimizing temperature and duration of Perm buffer for enhanced Intracellular Staining of Non-Human Primates Granulocyte Populations in flow cytometry

Author: Khushbu Komal, Flow Contract Site Laboratory, LLC

Abstract: This study investigates the optimization of intracellular staining protocols for flow cytometry in non-human primate (NHP) samples, with particular emphasis on the granulocyte population. It was noticed that perm buffer temperature and fixation time play a crucial role in preserving cell morphology. The standard protocol optimized for intracellular staining of human cells, the lysing and fixation steps resulted in the loss of the granulocyte population in NHP whole blood samples. To address this issue, modifications were introduced by experimenting with different perm buffer temperatures and incubation times. Through systematic adjustments, it was determined that utilizing a cold perm buffer with corresponding cold temperature incubation yielded the best results in terms of maintaining the granulocyte population in NHP specimens. Furthermore, the study addresses the potential implications of fixation conditions on reliability and reproducibility of flow cytometry data, with specific focus on minimizing variability in the granulocyte populations. The findings contribute to the establishment of standardized protocol for intracellular staining of NHP samples, ensuring robust and reproducible flow cytometry data, particularly in the context of studying granulocyte dynamics

Title: Application of Cell Painting Techniques Using an Imaging Flow Cytometer

Author: Alex Sutton, Cytek Biosciences

Abstract: Cell Painting is a method for staining critical cell organelles to establish a standard phenotype profile for each cell type and can be used to identify normal cells from those responding to drug treatments. The advantage of this approach is that it uses a standard “cell painting” protocol without the need to develop novel assays for each new drug being tested. Fluorescent dyes are applied to cells to ‘paint’ various cellular components, including but not limited to: DNA, mitochondria, the endoplasmic reticulum and Golgi apparatus, and the cytoskeleton. Cells can then be treated with chemical or genetic perturbagens and images analyzed for morphological changes. This allows for the collection of a large number of morphological measurements on a single cell. The current method for acquiring data for the Cell Painting assay is generally through High-Content Screening (HCS); however, this method is typically applied to adherent cells in a plate-based assay. This Cell Painting method can be adopted for cells in suspension using the Cytek® Amnis® ImageStream®X Mk II system. The Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer combines the high throughput performance of a flow cytometer with the imagery and functional insights of microscopy, and can be used to study the cell behaviors of adherent and suspension cells. In this study, we optimized the Cell Painting assay for cells in suspension using SYTO14 (nucleoli/RNA), MitoTracker Orange (mitochondria), Concanavalin A (endoplasmic reticulum), Wheat Germ Agglutinin (Golgi plasma membrane), Phalloidin (actin filaments), and Hoechst (DNA) to identify various cellular components and then used image analysis software to quantify the cell morphology and establish the cells’ phenotype profile. This cell profile can then be used to determine if cells respond to various treatments by changing their morphology. Through drug discovery, the Cell Painting assay and the Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer can help uncover targeted therapeutic effects of countless compounds using cells in suspension.

Title: Single Vesicle Flow Cytometry Using Cytek® Biosciences Flow Cytometry Systems

Author: Maria Gracia Garcia Mendoza, Cytek Biosciences

Abstract: Introduction: Extracellular vesicles (EVs) are cell-derived, membrane-bound small particles less than a micron in size. EVs are potentially valuable biomarkers and therapeutic agents in many disease environments, including cancer, autoimmunity, and neurodegenerative disorders. Due to their small size and heterogeneity, EVs can be difficult to characterize. Small particle/vesicle flow cytometry is a method developed with rigor and reproducibility capable of single EV measurements. Methods: Cytek® Aurora™ instruments was outfitted with the Enhanced Small Particle (ESP™) Detection option to increase the side scatter sensitivity. Amnis® ImageStream®x MKII was equipped with a 400 mW 488 nm laser and High Gain for added small particle sensitivity. Cytek systems were qualified using a set of hard-dyed beads (vCal™ nanoRainbow beads, Cellarcus Biosciences, Inc.) and calibrated with antibody capture beads (vCal™ nanoCal beads, Cellarcus Biosciences, Inc.) and a synthetic vesicle size standard (Lipo100TM, Cellarcus Biosciences, Inc.), followed by the conversion of fluorescence measurements to calibrated units for reporting. The multicolor vFC™ assay (Cellarcus Biosciences, Inc.) was performed according to the manufacturer’s workflows and protocols, using reference EVs, HEK 293T-derived recombinant EVs (Sigma), platelet-derived EVs (Cellarcus Biosciences, Inc.) and relevant positive and negative controls, as recommended by the MIFlowCyt-EV guidelines. Results: Our results demonstrate that Cytek flow cytometers can detect small particles. We were able to characterize particle size and surface cargo molecules, such as Tetraspanin expression and markers representative of the cell-of-origin. We can report the data with appropriate controls and in calibrated units, which are important for data publishing at the peer-review level. Conclusions: Here, we present a comprehensive workflow to analyze EVs, which includes instrument qualification, fluorescent channel calibration, sizing, and surface cargo measurements. These data help to address the requirements for improved standardization in reporting and reproducibility of EV flow cytometry data produced by spectral and imaging flow cytometry.

Title: A 30-color Immunophenotyping panel of Mice Infected with Influenza Using an Agilent NovoCyte Penteon Flow Cytometer

Author: HaiGuang Zhang, Agilent Technologies

Abstract: Use of flow cytometry has become routine across scientific disciplines, encompassing both the basic and clinical research spaces. It has proven to be a powerful tool to analyze different subpopulations of immune cells and gather a comprehensive overview of the immune system. Due to great advancements within the field, newer flow cytometers can support multiple colors, enabling identification of a variety of cells simultaneously. In this application note, a 30-color immunophenotyping panel was designed for the five-laser Agilent NovoCyte Penteon flow cytometer to see the distribution of immune cells in the lung, spleen, and mediastinal lymph nodes of mice infected with influenza.

Author: Kathy Bonness, PhD, BioLegend/Revvity

Title: Optimized Multicolor Flow Cytometry Panels

Abstract: Our expertly designed optimized flow cytometry panels take the guesswork out of panel and experimental planning, allowing you to focus on your next discovery. Our panel bundles include everything you need including fluorophore-conjugated antibodies, buffers, and a detailed protocol. Learn more about our optimized panels at

Title: Evaluating the Ability of the BD FACS Discover S8 to Sort Fragile Cells

Author: Nate Colven, Fred Hutchinson Cancer Center

Abstract: Cell sorting is an invaluable tool for researchers that is not without its downsides. When new products become available to the community, it is crucial that they are evaluated for how they compare to their competitors. The new BD Discover S8 FACS sorter features imaging technology that captures each cell the moment before it is sorted, giving cytometrists an unprecedented ability to evaluate how the stresses of cell sorting affect their cells. While sorting has been shown to minimally affect a cell’s proteomics and genomics, the stressors put upon a cell during sorting can lead to a decrease of metabolic function, increased stress response, and cell death (Ryan et. al. 2021). Many researchers use sorting as an intermediate step prior to culturing or injection into murine specimens and cell health and function is a key factor for these studies. To evaluate how the Discover S8 impacts sorting outcomes, we stained term placenta cells for cell function and nuclear heath. We then sorted out the cytotrophoblasts and macrophages and evaluated their health immediately post-sort and the morning after using confocal microscopy, comparing them to unsorted cells and cells sorted on a Miltenyi Tyto MACS sorter. These large, fragile cells had been unsuccessfully sorted on a BD Aria II and successfully sorted on the Tyto sorter in prior attempts. We found that when sorted on the Discover S8 a sufficient number of these cells survived to be used for downstream study.

Title: Tregs co-expressing ICOS and IL1R1 are enriched and highly suppressive within the human tumor microenvironment

Author: Andrew Konecny, Fred Hutchinson Cancer Center

Abstract: Complications from immunotherapies arise out of the pathways being targeted existing in nonmalignant tissue environments. A current gap in knowledge exists in understanding which immune alterations are unique for tumor maintenance and those that are characteristic of generic tissue inflammation. To determine immunological changes unique to the tumor microenvironment, we compared human head neck squamous cell carcinoma to site matched inflamed non-malignant oral mucosa using several single cell and computational approaches. In addition to finding considerable overlap between the immune landscapes of nonmalignant and tumor specific inflammation, we found that Tregs co-expressing ICOS and the IL-1 receptor type 1 (IL1R1) to be highly enriched and unique to the tumor microenvironment. Through continued investigation, we provide evidence that IL1R1+ Tregs have recently encountered antigen, have undergone clonal expansion, and are transcriptionally and functionally unique when compared to IL1R1- Tregs. Additionally, we found IL1R1+ Tregs in the tumor-draining lymph nodes. We are currently investigating the relationship of IL1R1+ Tregs between the tumor and lymph node with the goal of determining where these IL1R1+ Tregs are generated.

Title: Why Use Cytek Aurora & Aurora CS Systems?

Author: Jessi Tuengel, PhD, Cytek Bio

Abstract: The Cytek Aurora™ cell analyzer and the Cytek Aurora™ CS system are both built on the same paradigm-shifting Full Spectrum Profiling™ (FSP™) technology that enables scientists to distinguish fluorochromes with highly overlapping peak emissions, increasing fluorochrome choice and multiplexing capability. Data clarity on highly autofluorescent samples is further improved with autofluorescence characterization and extraction. Panels acquired on the Aurora can be seamlessly transferred to the Aurora CS sorter with comparable high resolution and without the need to reconfigure the experiment.