Flow Cytometric Identification Research Articles

Isolation and high-dimensional flow cytometric analysis of tumor-infiltrating leukocytes in a mouse model of colorectal cancer.

Colorectal cancer (CRC) is a complex and heterogeneous disease characterized by dysregulated interactions between tumor cells and the immune system. The tumor microenvironment plays a pivotal role in cancer initiation as well as progression, with myeloid immune cells such as dendritic cell and macrophage subsets playing diverse roles in cancer immunity. On one hand, they exert anti-tumor effects, but they can also contribute to tumor growth. The AOM/DSS colitis-associated cancer mouse model has emerged as a valuable tool to investigate inflammation-driven CRC. To understand the role of different leukocyte populations in tumor development, the preparation of single cell suspensions from tumors has become standard procedure for many types of cancer in recent years. However, in the case of AOM/DSS-induced colorectal tumors, this is still challenging and rarely described. For one, to be able to properly distinguish tumor-associated immune cells, separate processing of cancerous and surrounding colon tissue is essential. In addition, cell yield, due to the low tumor mass, viability, as well as preservation of cell surface epitopes are important for successful flow cytometric profiling of tumor-infiltrating leukocytes. Here we present a fast, simple, and economical step-by-step protocol for isolating colorectal tumor-associated leukocytes from AOM/DSS-treated mice. Furthermore, we demonstrate the feasibility of this protocol for high-dimensional flow cytometric identification of the different tumor-infiltrating leukocyte populations, with a specific focus on myeloid cell subsets.

Computational Flow Cytometry Accurately Identifies Sezary Cells Based on Simplified Aberrancy and Clonality Features

Flow cytometric identification of circulating neoplastic cells (Sezary cells) in patients with mycosis fungoides (MF) and Sezary syndrome (SS) is essential for diagnosis, staging and prognosis. While recent advances have improved the performance of this laboratory assay, the complex immunophenotype of Sezary cells and overlap with reactive T cells demand a high level of analytic expertise. We utilized machine learning to simplify this analysis using only 2 pre-defined Sezary cell-gating plots. We studied 114 samples from 59 patients with SS/MF, and 66 samples from unique patients with inflammatory dermatoses. A single dimensionality reduction plot highlighted all T-cell receptor constant β chain-restricted (clonal) CD3+/CD4+ T-cells detected by expert analysis. On receiver operator curve analysis, an aberrancy scale feature computed by comparison with controls (area under the curve = 0.98) outperformed loss of CD2 (0.76), CD3 (0.83), CD7 (0.77) and CD26 (0.82) in discriminating Sezary cells from reactive CD4+ T cells. Our results closely mirrored those obtained by exhaustive expert analysis for event classification (positive percent agreement = 100%, negative percent agreement = 99%) and Sezary cell quantitation (regression slope = 1.003, R squared = 0.9996). We demonstrate the potential of machine learning to simplify the accurate identification of Sezary cells.

Flow Cytometric Identification of Human IgE+ B Lineage Subsets.

The use of flow cytometry for immunophenotyping is contingent on the ability to accurately assign biological relevance to the detected signal. This process has historically been challenging when defining IgE expressing B cells or IgE expressing antibody-secreting cells due to widespread expression of receptors for IgE on various leukocyte subsets, including human B cells. Here we describe our implementation of intracellular staining for human IgE following a blocking step to negate the challenge of surface-bound IgE. We also describe our experience with a human B cell culture system that can be used to robustly validate this approach before application to primary human samples. Orthogonal confirmatory techniques remain essential; these are not described in detail, but several possible strategies are suggested.

A Flow Cytometry Study of the Binding and Stimulation Potential of Inactivated Trypanosoma evansi toward Dromedary Camel Leukocytes.

Surra, a wasting disease caused by Trypanosoma evansi, is one of the major animal health burdens in camel-rearing countries, imposing significant economic losses due to reduced fertility and high mortality rates. The present study used inactivated T. evansi (from the Card Agglutination Test for Trypanosomes/Trypanosoma evansi; CATT/T. evansi) and flow cytometry to investigate their binding and activation potential toward camel leukocyte subsets. Labeling T. evansi with propidium iodide (PI) enabled their flow cytometric enumeration and identification with forward scatter (FSC; indicative for cell size) and side scatter (SSC; indicative for cell internal complexity) characteristics that are comparable with values reported for Trypanosoma cruzi. The incubation of PI-labeled non-opsonized T. evansi with camel leukocyte populations revealed that camel monocytes have the highest potential to bind T. evansi, followed by granulocytes and lymphocytes. The identification of pattern recognition receptors (PRRs) on camel immune cells and the pathogen-associated molecular patterns (PAMPs) in T. evansi that are responsible for this different binding capacity requires further studies. Stimulation of camel neutrophils with Trypanosoma evansi induced shape change, reactive oxygen species (ROS) production, and neutrophil extracellular traps (NET)-formation. To ensure that T. evansi, in the parasite concentration used in this study, is not apoptotic or necrotic to camel leukocytes, we evaluated cell apoptosis and necrosis after stimulation with T. evansi. The results revealed no impact of T. evansi stimulation for 2 h on the cell viability of camel leukocytes. Subsequent work may focus on the diagnostic employment of labeled T. evansi and flow cytometry for the detection of anti-Trypanosoma antibodies in camel serum. In addition, more efforts should be deployed to investigate the host-pathogen interaction mechanisms and the escape mechanisms of T. evansi in camels. To complete these data, further studies using the living or freshly killed parasites could also be implemented in camels and/or horses.

Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits

To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.

Flow cytometric detection and identification of different leukocyte subpopulations in stallion semen.

To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups, 27.85 ± 5.7% of events were positive for CD4, CD8, CD21 or CD172a, while in native semen samples, leukocytes were scarce (0.114 ± 0.134%). The most abundant leukocyte subpopulation in semen was of lymphoid origin (CD4-positive cells [0.015 ± 0.02%]), whereas CD21-positive cells (B cells; 0.001 ± 0.001%) were virtually absent in ejaculates of fertile stallions. This presented flow cytometric assay for the detection and identification of different leukocyte population in equine antioxidant-treated ejaculates can be used as an additional tool for spermatological examination in stallions.

COVID-19 Adenoviral Vector Vaccination Elicits a Robust Memory B Cell Response with the Capacity to Recognize Omicron BA.2 and BA.5 Variants.

Following the COVID-19 pandemic, novel vaccines have successfully reduced severe disease and death. Despite eliciting lower antibody responses, adenoviral vector vaccines are nearly as effective as mRNA vaccines. Therefore, protection against severe disease may be mediated by immune memory cells. We here evaluated plasma antibody and memory B cells (Bmem) targeting the SARS-CoV-2 Spike receptor-binding domain (RBD) elicited by the adenoviral vector vaccine ChAdOx1 (AstraZeneca), their capacity to bind Omicron subvariants, and compared this to the response to mRNA BNT162b2 (Pfizer-BioNTech) vaccination. Whole blood was sampled from 31 healthy adults pre-vaccination and 4weeks after dose one and dose two of ChAdOx1. Neutralizing antibodies (NAb) against SARS-CoV-2 were quantified at each time point. Recombinant RBDs of the Wuhan-Hu-1 (WH1), Delta, BA.2, and BA.5 variants were produced for ELISA-based quantification of plasma IgG and incorporated separately into fluorescent tetramers for flow cytometric identification of RBD-specific Bmem. NAb and RBD-specific IgG levels were over eight times lower following ChAdOx1 vaccination than BNT162b2. In ChAdOx1-vaccinated individuals, median plasma IgG recognition of BA.2 and BA.5 as a proportion of WH1-specific IgG was 26% and 17%, respectively. All donors generated resting RBD-specific Bmem, which were boosted after the second dose of ChAdOx1 and were similar in number to those produced by BNT162b2. The second dose of ChAdOx1 boosted Bmem that recognized VoC, and 37% and 39% of WH1-specific Bmem recognized BA.2 and BA.5, respectively. These data uncover mechanisms by which ChAdOx1 elicits immune memory to confer effective protection against severe COVID-19.

Single-Cell Techniques in Environmental Microbiology

Environmental microbiology has been an essential part of environmental research because it provides effective solutions to most pollutants. Hence, there is an interest in investigating microorganism behavior, such as observation, identification, isolation of pollutant degraders, and interactions between microbial species. To comprehensively understand cell heterogeneity, diverse approaches at the single-cell level are demanded. Thus far, the traditional bulk biological tools such as petri dishes are technically challenging for single cells, which could mask the heterogeneity. Single-cell technologies can reveal complex and rare cell populations by detecting heterogeneity among individual cells, which offers advantages of higher resolution, higher throughput, more accurate analysis, etc. Here, we overviewed several single-cell techniques on observation, isolation, and identification from aspects of methods and applications. Microscopic observation, sequencing identification, flow cytometric identification and isolation, Raman spectroscopy-based identification and isolation, and their applications are mainly discussed. Further development on multi-technique integrations at the single-cell level may highly advance the research progress of environmental microbiology, thereby giving more indication in the environmental microbial ecology.

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood.

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++ CD16-/low ), intermediate (intM; CD14++/+ CD16+ ), and non-classical (ncM; CD14-/low CD16++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++ /CD16+ . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies.

Flow Cytometric Identification of Hematopoietic and Leukemic Blast Cells for Tailored Clinical Follow-Up of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a myeloid malignancy that is characterized by the accumulation of leukemic blast cells, which originate from hematopoietic stem cells that have undergone leukemic transformation and/or are more mature progenitors that have gained stemness features. Currently, no consensus exists for the flow cytometric identification of normal blast cells and their leukemic counterparts by their antigenic expression profile. Differentiating between the benign cells and the malignant cells is crucial for the further deployment of immunophenotype panels for the clinical follow-up of AML patients. This review provides an overview of immunophenotypic markers that allow the identification of leukemic blast cells in the bone marrow with multiparameter flow cytometry. This technique allows the identification of hematopoietic blast cells at the level of maturing cells by their antigen expression profile. While aberrant antigen expression of a single immunophenotypic marker cell cannot be utilized in order to differentiate leukemic blast cells from normal blast cells, combinations of multiple immunophenotypic markers can enable the distinction of normal and leukemic blast cells. The identification of these markers has provided new perspectives for tailored clinical follow-up, including therapy management, diagnostics, and prognostic purposes. The immunophenotypic marker panels, however, should be developed by carefully considering the variable antigen marker expression profile of individual patients.

Identification of peripheral blood CD26+ leukemic stem cells has a potential role in the rapid diagnosis of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (SC) neoplasm diagnosed by the demonstration of t(9;22)(BCR-ABL1) fusion gene. We performed a flow cytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for CML and its utility for detection of residual disease in CML patients on therapy. Patients with clinical suspicion of CML/CML on follow-up were included, and peripheral (PB) and/or bone marrow (BM) samples were utilized for flow cytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. A pre-titrated antibody cocktail containing CD45, CD34, CD38, and CD26MoABs was used. A total of 104samples (63PB and 41 BM) from 64 patients [suspected CML (n=30), CML on follow-up (n=15), and non-CML (n=19)] were tested. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (median=0.07 (range 0.002%-26.79%)). None of the patients in the control group (non-CML) and follow-up patients with negative reverse transcriptase-polymerase chain reaction (RT-PCR) results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r=.917). Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust, and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is irrespective of BCR-ABL1 transcript type, and its role in residual disease monitoring needs thorough investigation.

Flow Cytometry Based Identification of CD26+ Leukemic Stem Cells in the Peripheral Blood Has a Potential Role in the Rapid Diagnosis of Chronic Myeloid Leukemia

Background: Demonstration of t(9;22)(BCR-ABL1) fusion is gold standard for the diagnosis of chronic myeloid leukemia (CML). We performed a flowcytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for the diagnosis of CML and its utility for detection of residual disease in CML patients on therapy.Methods: Patients of CML/ CML on follow-up were included and peripheral (PB) and/or bone marrow (BM) samples were utilized for flowcytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. Under ‘lyse-wash-stain-wash’ sequence, the sample was incubated with a pre-titrated custom-made antibody cocktail in a ‘test’ tube containing CD45, CD34, CD38 and CD26 mo-abs. Acquisition was carried out on BD FACS Canto II and analysis was done with Diva Software. Clinical data including demographic details, complete blood count and BM findings were also noted.Results: A total of 104 samples (63 PB and 41 BM) from 64 patients [confirmed & treatment naïve CML (n=30), CML on follow-up (n=15), non-CML (n=19)] were tested. The median (range) time for reporting of PB/BM examination, molecular genetic studies and flow cytometry for CD26+ CML LSCs was 5 (3-11 days), 4 (3-6 days) and 1 (0-1 day) respectively. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (Median=0.07%, range 0.002%-26.79%), and also in 8/15 patients of the follow-up group, who also reported persisting levels of BCR-ABL1. None of the patients in the non-CML group and follow-up CML patients with negative RT-PCR results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r=0.917).Conclusion: Flow cytometric assessment for CD26+ LSCs is quick with reporting time of even less than an hour. Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is independent of BCR-ABL1 transcript type and its role in residual disease monitoring needs further investigation. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometric Quantification of Cytotoxic Activity in Whole Blood Samples.

Current methods for the determination of cell-mediated cytotoxic activity in blood samples usually isolate peripheral blood mononuclear cells by density gradient centrifugation or alternatively use erythrocyte lysis. Both centrifugation and red cell lysis can cause cellular depletion and cell dysfunction, resulting in erroneous measurements. To address limitations of current assays, we developed an improved strategy to determine cellular cytotoxicity using flow cytometry. Viable nucleic acid stains are used to identify live nucleated cells and discriminate them from non-nucleated erythrocytes, platelets, and debris while avoiding lysing and washing steps to maintain cell functionality. To detect target cells, we have used two different labeling approaches. In the first approach, EGFP-labeled K562 human chronic myelogenous leukemia cells provide a "ready-to-use" target without the need of additional for labeling or staining. For the second approach, we perform parallel cytotoxicity assays in the presence of wild-type K562 cells previously loaded with a fluorescent dye that has spectral properties similar to those of EGFP. Given the importance of cytotoxic assays and the deleterious effects of current sample preparation methods, the aim of this study was to adapt this "untouched cells" flow cytometry method to study cytotoxic activity using unlysed whole blood samples and fluorescent target cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for cell-mediated cytotoxic activity determination in unlysed whole blood Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of cell-mediated cytotoxic activity using unlysed whole blood samples Support Protocol 1: Optimization of the performance of target cell labeling approaches Support Protocol 2: Assessment of the linearity and reproducibility of cytotoxicity assays.

Role of Flow Cytometric Immunophenotyping for Classic Hodgkin Lymphoma in Small Biopsy and Cytology Specimens.

The diagnosis of classic Hodgkin lymphoma (CHL) traditionally requires surgical tissue biopsy because of the paucity of diagnostic Hodgkin and Reed-Sternberg cells. Diagnosis can be challenging in small core needle and cytologic biopsies, which are increasingly used because of reduced costs and minimal invasiveness. Flow cytometric (FC) identification of Hodgkin and Reed-Sternberg cells is possible, but FC test efficacy is not well studied outside of validation settings, especially in small specimens. To assess the testing efficacy of FC performed on small biopsy and cytology specimens for the diagnosis of CHL. We reviewed 131 patients with CHL and 459 patients without CHL during a 3-year period who underwent a small biopsy procedure, including core biopsy and/or cytology evaluation, with concurrent routine clinical FC testing for CHL, assessing performance of FC in small specimens. Evaluating testing efficacy, sensitivity was 95.4% and specificity was 98.2%, whereas positive and negative predictive values were 92.2% and 99.0%, respectively. Although there were more false-positive results than compared with published validation studies, expert review identified distinct diagnostic pitfalls; awareness of these may improve testing efficacy. Although FC diagnosis of CHL was historically considered unfeasible, our findings in a real-world clinical setting suggest that FC adds diagnostic value to small biopsy evaluation, reducing time to treatment, costs, and invasive excisional procedures.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Comprehensive phenotyping of endothelial cells using flow cytometry 1: Murine.

The endothelium forms a selective barrier between circulating blood or lymph and surrounding tissue. Endothelial cells play an essential role in vessel homeostasis, and identification of these cells is critical in vascular biology research. However, characteristics of endothelial cells differ depending on the location and type of blood or lymph vessel. Endothelial cell subsets are numerous and often identified using different flow cytometric markers, making immunophenotyping these cells complex. In part 1 of this two part review series, we present a comprehensive overview of markers for the flow cytometric identification and phenotyping of murine endothelial subsets. These subsets can be distinguished using a panel of cell surface and intracellular markers shared by all endothelial cells in combination with additional markers of specialized endothelial cell types. This review can be used to determine the best markers for identifying and phenotyping desired murine endothelial cell subsets.

Flow Cytometric Quantification of Granulocytic Alkaline Phosphatase Activity in Unlysed Whole Blood.

Translational research has improved the diagnosis and follow-up of hematological diseases and malignancies. However, some classical diagnostics used for research and clinical practice that have remain practically unchanged for decades may be better addressed through advances in flow cytometry technology, whereby more precise measurements may be implemented in a straightforward manner. The current method for semiquantitative analysis of granulocytic alkaline phosphatase (GAP) activity is still based on observer-dependent color-intensity classification. Here, we describe a novel strategy for flow cytometric quantification of GAP activity in which staining and analytical flow cytometry facilitate the detection and quantification of subpopulations of leukocytes with different GAP activities. Our experiments demonstrate the potential of flow cytometry as a simple and highly sensitive approach for measuring GAP activity in unlysed whole blood. Notably, a comparison of flow cytometry and enzyme cytochemistry techniques showed that enzyme activity scores were not similar, indicating that results needs to be interpreted with caution, given that the enzyme-substrate binding affinities may differ, as well as the subjective evaluation of the intensity of the precipitated dye. © 2020 Wiley Periodicals LLC. Basic Protocol: Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of alkaline phosphatase activity in granulocytes from whole blood samples Support Protocol 1: Sample preparation for granulocyte alkaline phosphatase determination by flow cytometry using no-lyse no-wash methods Support Protocol 2: Data analysis and formula to calculate the GAP score.

Flow cytometric identification and cell-line establishment of macrophages in naked mole-rats

Naked mole rats (NMRs) have extraordinarily long lifespans and anti-tumorigenic capability. Recent studies of humans and mice have shown that many age-related diseases, including cancer, are strongly correlated with immunity, and macrophages play particularly important roles in immune regulation. Therefore, NMR macrophages may contribute to their unique phenotypes. However, studies of the roles of macrophages are limited by material restrictions and the lack of an established experimental strategy. In this study, we developed a flow cytometric strategy to identify NMR macrophages. The NMR macrophages were extractable using an off-the-shelf anti-CD11b antibody, M1/70, and forward/side scatter data obtained by flow cytometry. NMR macrophages proliferated in response to human/mouse recombinant M-CSF and engulfed Escherichia coli particles. Interestingly, the majority of NMR macrophages exhibited co-staining with an anti-NK1.1 antibody, PK136. NK1.1 antigen crosslinking with PK136 results in mouse NK cell stimulation; similarly, NMR macrophages proliferated in response to NK1.1 antibody treatment. Furthermore, we successfully established an NMR macrophage cell line, NPM1, by transduction of Simian virus 40 early region that proliferated indefinitely without cytokines and retained its phagocytotic capacity. The NPM1 would contribute to further studies on the immunity of NMRs.

Feasibility of Flow Cytometric Identification and Isolation of Pre-Platelets to Understand Its Function and Role in Cancer Metastasis

Background:It is known that platelets interact with tumors to support malignant cell growth and stimulate epithelial to mesenchymal transition (Li, et al. "The Role of Platelets in Tumor Growth, Metastasis, and Immune Evasion", Platelets, pp. 547-561, Academic Press, 2019). P-selectin which is secreted from alpha granules has been implicated as the major perpetrator of this tumor cell and platelet communication. (Kim et al, PNAS 95(16): 9325-9330, 1998). Higher numbers of alpha granules are found in newly released young platelets from megakaryocytes, also known as pre-platelets, than mature platelets. Given their potential role in cancer metastasis, the ability to identify and isolate pre-platelets from mature plateletsforassessment of overall functionality and biology is important. Of note, identification and measurement of pre-platelets based on higher RNA content has been in clinical use for various clinical conditions for several years. Here we evaluated the feasibility of identifying and sorting pre-platelets by flow cytometric analyses in multiple platelet apheresis samples from heathy donors. Study Design and Methods Apheresis samples from adult healthy donors at Roswell Park Comprehensive Cancer Center were obtained and analyzed in accordance with an IRB approved protocol. Platelets sampled from apheresis products were collected with the use of an apheresis device known to yield a leuko-reduced platelet concentrate. All components were irradiated with 2500 cGy immediately after the collection and stored on platelet agitator at 22°C. A 4 mL aliquot was withdrawn from each bag for sorting essays.Pre-platelets were distinguished from mature platelets based on the presence of higher RNA levels. The cell permeant RNA dye TO (BD Biosciences, San Jose, CA) was used to identify pre-plapletes by flow cytometry according to published protocol (Ando et al Blood97 (4): p915-921, 2001).The optimal concentration of TO dye was determined by titration at decreasing dilutions and acquisition on a LSR-II flow cytometer (BD Biosciences, San Jose, CA). Confirmation of flow cytometry-based assay was determined by processing 5 healthy donor whole blood samples by both flow cytometry and using an XE-5000 automated hematology analyzer (Sysmex America, Mundelein, IL). The percentage of pre-platelets was measured as a percent of total platelets by both techniques. Platelet sorting was performed on 12 leuko-reduced apheresis platelet products on a FACSAria II (BD Biosciences, San Jose, CA). Platelets were identified by co-staining with CD41a. Platelet activation was assessed before and after sorting based on P-selectin (CD62P) surface expression. Flow cytometry analysis was performed using FCSExpress v6. Results: We determined that flow cytometric sorting of pre-platelets optimally required TO (0.5 µg/mL) for optimal staining for gating of pre-platelets. A two way, paired simple t test resulted in a p value of 0.09 when compared to standard gating strategy per XE-5000 automated hematology analyzer. Among all 14 samples evaluated, the percentage of preplatelet to apheresis sample pre-platelet ratio was estimated between 2.8% to 8.2%. Interestingly P-selectin ratio of pre-platelets was significantly higher than mature platelets (median: 67% vs 52%). ConclusionThis study is the first to demonstrate the feasibility of identifying and sorting pre-platelets using a novel flow cytometric method with TO staining. Isolation of these rare platelet precursor cells (pre-platelets) with known higher P-selectin expression are an essential first step to understanding the potential pre-eminent role of these cells in cancer evasion and metastasis. Disclosures Wang: Abbvie: Other: Advisory role; Kite: Other: Advisory role; Jazz: Other: Advisory role; Astellas: Other: Advisory role, Speakers Bureau; celyad: Other: Advisory role; Pfizer: Other: Advisory role, Speakers Bureau; Stemline: Other: Advisory role, Speakers Bureau; Daiichi: Other: Advisory role; Amgen: Other: Advisory role; Agios: Other: Advisory role.

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy

R. Jimenez-Saiz, Y. Ellenbogen and K. Bruton et al. developed a method to identify bona fide IgE+ MBCs in humans, demonstrated their extreme rarity in circulation and cautioned against the clinical utility of their assessment.