Isolation Of Immune Cells Research Articles

Development of Ex Vivo Analysis for Examining Cell Composition, Immunological Landscape, Tumor and Immune Related Markers in Non-Small-Cell Lung Cancer.

NSCLC is a very aggressive solid tumor, with a poor prognosis due to post-surgical recurrence. Analysis of the specific tumor and immune signatures of NSCLC samples is a critical step in prognostic evaluation and management decisions for patients after surgery. Routine histological assays have some limitations. Therefore, new diagnostic tools with the capability to quickly recognize NSCLC subtypes and correctly identify various markers are needed. We developed a technique for ex vivo isolation of cancer and immune cells from surgical tumor and lung tissue samples of patients with NSCLC (adenocarcinomas and squamous cell carcinomas) and their examination on ex vivo cell preparations and, parallelly, on histological sections after Romanovsky-Giemsa and immunofluorescent/immunochemical staining for cancer-specific and immune-related markers. As a result, PD-L1 expression was detected for some patients only by ex vivo analysis. Immune cell profiling in the tumor microenvironment revealed significant differences in the immunological landscapes between the patients' tumors, with smokers' macrophages with simultaneous expression of pro- and anti-inflammatory cytokines, neutrophils, and eosinophils being the dominant populations. The proposed ex vivo analysis may be used as an additional diagnostic tool for quick examination of cancer and immune cells in whole tumor samples and to avoid false negatives in histological assays.

Effects of general anesthesia on airway immune cell function in an equine in vivo model

ObjectiveComplications from general anesthesia, including pneumonia and decreased wound healing, are influenced by changes in immune cell function secondary to sedatives and anesthetics. It was hypothesized that immune cell function would be depressed in the early postanesthetic period. The objective was to investigate airway immune cell function before and after a general anesthetic episode in an equine in vivo model using ex vivo cell stimulations with lipopolysaccharide (LPS) for assessment of immune function. Study designProspective experimental study. AnimalsSix healthy, adult, institution-owned horses. MethodsEach horse underwent a bronchoalveolar lavage (BAL) 3 days before and immediately after a 2 hour general anesthetic. The BAL fluid was examined for cytology, total nucleated cell count and isolation of immune cells. Airway immune cells were treated with LPS or media (control) for 6 hours and supernatant was analyzed via a commercially available immunoassay for cytokines [tumor necrosis factor alpha (TNFα), interleukin (IL)-1β, IL-6, interferon gamma (IFNγ) and CXC motif chemokine ligand 8 (CXCL8)]. Data were compared using t-tests and Mann–Whitney tests. ResultsBefore anesthesia (baseline), LPS stimulation induced a significant increase in all cytokines of interest, except CXCL8, versus control samples. Unstimulated cells, after an anesthetic episode, had a significant 1.8-fold increase in IL-1β (p = 0.029), and a significant decrease in IL-6 and TNFα (p = 0.028 and 0.033, respectively) versus baseline. Following anesthesia, stimulated cells had a significant decrease in IL-6 and TNFα (p = 0.037 and 0.042, respectively) versus baseline. Conclusions and clinical relevanceThis study supports the use of an equine in vivo model to assess airway immune cell function in relation to general anesthetic use.

Inclusion of Lacticaseibacillus paracasei NSMJ15 in broiler diets induces changes in jejunal immune cell population and cecal microbiota

Objective: The objective was to investigate growth performance, antioxidant enzyme activity, intestinal morphology, immune cell distribution, short chain fatty acid (SCFA) profile, and microbiota in broiler chickens fed a diet containing <i>Lacticaseibacillus paracasei</i> NSMJ15.Methods: A total of 120 1-day-old Ross 308 male broilers were allocated to 2 dietary treatments in a randomized complete block design. A control group was fed a corn-soybean meal control diet, and an NSMJ15-supplemented group was fed a control diet supplemented with 1 g/kg <i>L. paracasei</i> NSMJ15 at the expense of cornstarch. Each dietary treatment had 6 replicates with 10 birds per cage. Growth performance was recorded on day 9. On day 10, one bird representing median body weight was selected to collect serum for antioxidant enzyme activity, jejunal tissue for immune cell isolation and morphometric analysis, and cecal digesta for 16S rRNA gene sequencing and SCFA analysis.Results: Supplementation of <i>L. paracasei</i> NSMJ15 did not affect growth performance, serum antioxidant enzyme activity, and jejunal histomorphology compared to the control group. In the NSMJ15-supplemented group, the population of CD3+CD4+CD8− T cells increased (p = 0.010), while the population of CD3+CD8+TCRγδ+ T cells decreased (p = 0.022) compared to the control group. The <i>L. paracasei</i> NSMJ15 supplementation decreased (p = 0.022) acetate concentration in the cecal digesta compared to the control group. The 16S rRNA gene sequencing analysis showed that NSMJ15-supplemented group differentially expressed (p<0.05) 10 more amplicon sequence variants compared to control group without affecting alpha and beta diversity indices of the cecal microbiota. Genera <i>Mediterraneibacter</i> and <i>Negativibacillus</i> were positively (p<0.05) correlated with CD4+ T cells, while genera <i>Gemmiger</i>, <i>Coprococcus</i>, <i>Sellimonas</i>, <i>Massilimicrobiota</i>, and <i>Blautia</i> were negatively (p<0.05) correlated with SCFA concentration.Conclusion: The results of the present study suggest dietary <i>L. paracasei</i> NSMJ15 supplementation may increase percentage of CD4+ T cells and decrease acetate concentration in broiler chickens by increasing the differential expression of specific microbial genera.

Flow Cytometry Characterization and Analysis of Glial and Immune Cells from the Spinal Cord

Several protocols have been developed with the aim of characterizing glial and immune cells from the central and peripheral nervous systems. However, a small number of these protocols have demonstrated the ability to yield satisfactory results following conventional isolation. Considering this necessity and the difficulties encountered in enzymatic and bead isolation, our work proposes a method for the isolation of glial and immune cells from the spinal cord utilizing a Percoll gradient. For this purpose, C57BL/6J spinal cords were dissected, and the lumbar intumescence was dissociated and subjected to a Percoll gradient centrifugation (70%, 50%, 37%, and 10%). Each layer was then separated and labeled for astrocytes (anti-GFAP, TNF-α, IFN-γ, IL-10, IL-4), microglia (anti-CD45, CD11b, CD206, CD68, TNF-α, IFN-γ), and lymphocytes (anti-CD3, CD4, IFN-γ, IL-4). The gate detections were mathematically performed by computational analysis utilizing the K-means clustering algorithm. The results demonstrated that astrocytes were concentrated at the Percoll 10/37 interface, microglia at the Percoll 37/50 layer, and lymphocytes at the Percoll 50/70 layer. Our findings indicate that astrocytes in healthy animals are putative of the A1 profile, while microglia and lymphocytes are more frequently labeled with M1 and Th1 markers, suggesting a propensity towards inflammatory responses. The computational method enabled the semi-autonomous gate detection of flow cytometry data, which might facilitate and expedite the processing of large amounts of data.

Calorie restriction-induced leptin reduction and T-lymphocyte activation in blood and adipose tissue in men with overweight and obesity

BackgroundT-Lymphocyte activation is modulated by the adipokine leptin and serum concentrations of this hormone can be reduced with short-term calorie restriction. The aim of this study was to understand whether leptin per se is important in determining levels of T-lymphocyte activation in humans, by investigating whether the reduction in leptin concentration following calorie restriction is associated with a decrease in T-Lymphocyte activation in blood and adipose tissue.MethodsTwelve men with overweight and obesity (age 35–55 years, waist circumference 95–115 cm) reduced their calorie intake by 50% for 3 consecutive days. Blood and subcutaneous adipose tissue were obtained for isolation of immune cells and cytokine analysis. CD4+ and CD8 + T-Lymphocytes were identified and characterised according to their expression of activation markers CD25 and CD69 by flow cytometry.ResultsSerum leptin was reduced by (mean ± SEM) 31 ± 16% (p < 0.001) following calorie restriction. The percentage of blood CD4 + CD25 + T-lymphocytes and level of CD25 expression on these lymphocytes were significantly reduced by 8 ± 10% (p = 0.016) and 8 ± 4% (p = 0.058), respectively. After calorie restriction, ex vivo leptin secretion from abdominal subcutaneous adipose tissue explants was not changed, and this corresponded with a lack of change in adipose tissue resident T-Lymphocyte activation.ConclusionsSerum leptin was reduced after calorie restriction and this was temporally associated with a reduction in activation of blood CD4 + CD25 + T-Lymphocytes. In abdominal subcutaneous adipose tissue, however, leptin secretion was unaltered, and there were no observed changes in adipose resident T-Lymphocyte activation.

Isolation and functional identification of immune cells in hemolymph of blood clams Tegillarca granosa

Blood clam Tegillarca granosa is a type of economically cultivated bivalve mollusk with red blood, and it primarily relies on hemocytes in its hemolymph for immune defense. However, there are currently no reports on the isolation and identification of immune cells in T. granosa, which hinders our understanding of their immune defense. In this study, we employed single-cell transcriptome sequencing (scRNA-seq) to visualize the molecular profile of hemocytes in T. granosa. Based on differential expression of immune genes and hemoglobin genes, hemocytes can be molecularly classified into immune cells and erythrocytes. In addition, we separated immune cells using density gradient centrifugation and demonstrated their stronger phagocytic capacity compared to erythrocytes, as well as higher levels of ROS and NO. In summary, our experiments involved the isolation and functional identification of immune cells in hemolymph of T. granosa. This study will provide valuable insights into the innate immune system of red-blood mollusks and further deepen the immunological research of mollusks.

Isolation of Immune Regulatory Sertoli Cells from Mouse Seminiferous Tubules

Isolation of Immune Regulatory Sertoli Cells from Mouse Seminiferous Tubules

Isolation of Immune Regulatory Sertoli Cells from Mouse Seminiferous Tubules

Isolation of Immune Regulatory Sertoli Cells from Mouse Seminiferous Tubules

Murine Models of Salmonella Infection.

The pathogen Salmonella enterica encompasses a range of bacterial serovars that cause intestinal inflammation and systemic infections in humans. Mice are a widely used infection model due to their relative simplicity and versatility. Here, we provide standardized protocols for culturing the prolific zoonotic pathogen S. enterica serovar Typhimurium for intragastric inoculation of mice to model colitis or systemic dissemination, along with techniques for direct extraintestinal infection. Furthermore, we present procedures for quantifying pathogen burden and for characterizing the immune response by analyzing tissue pathology, inflammatory markers, and immune cells from intestinal tissues. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Murine colitis model utilizing oral streptomycin pretreatment and oral S. Typhimurium administration Basic Protocol 2: Intraperitoneal injection of S. Typhimurium for modeling extraintestinal infection Support Protocol 1: Preparation of S. Typhimurium inoculum Support Protocol 2: Preparation of mixed S. Typhimurium inoculum for competitive infection Basic Protocol 3: Assessment of S. Typhimurium burden Support Protocol 3: Preservation and pathological assessment of S. Typhimurium-infected tissues Support Protocol 4: Measurement of inflammatory marker expression in intestinal tissues by qPCR Support Protocol 5: Preparation of intestinal content for inflammatory marker quantification by ELISA Support Protocol 6: Immune cell isolation from Salmonella-infected intestinal tissues.

Erratum to “Isolation and purification of immune cells from the liver” [Int. Immunopharmacol. 85 (2020) 106632

Erratum to “Isolation and purification of immune cells from the liver” [Int. Immunopharmacol. 85 (2020) 106632

Abstract 2396: Longitudinal monitoring of stromal stiffness predicts immune escape in preclinical hepatocellular carcinoma

Abstract Background/Objectives: Increased tumor stiffness enhances disease progression and aggressiveness in several cancers. Stiffer extracellular tumor matrix hinders T cell trafficking and promotes immune escape. The interplay between liver tumor mechanical properties and hepatocellular immune cell landscape has not been fully investigated. The aim of our study is to correlate tumor stiffness quantified with shear wave elastography and tumor-infiltrating lymphocyte accumulation using a syngeneic orthotopic rat model of hepatocellular carcinoma (HCC). Methods: Rat hepatoma cell line McA-RH7777 were implanted in the left liver lobe of Buffalo rats. Maximum tumor size and stiffness were evaluated 3 weeks after implantation (baseline) and weekly for 14 days using ultrasound. The maximum tumor size of each rat was evaluated on transverse view of B-mode ultrasound. The elasticity of each rat was evaluated by shear wave elastography (SWE), and SWE measurements were obtained at the maximum tumor size in m/s. A circular region of interest was put manually on the SWE images to calculate tumor stiffness. SWE was performed 3 times for each rat at each time point, and the mean value of tumor stiffness was used for analysis. After 14 days, tumors were harvested for histology and immune cell isolation. Isolated cells were stained with CD3, CD4, CD8, CD25, CD45, CD103, CD161, CD279 (PD-1), FOXP3, IL17A, and INF-g and submitted to spectral cytometry. Results: Shear wave speed at baseline and at 14 days ranged from 1.95 to 3.07 m/s and from 2.25 to 3.26 m/s, respectively. The rate of longitudinal increase or decrease in tumor stiffness measured by SWE is inversely correlated with intratumoral infiltration of CD45+/CD3+, CD3+/CD8+ T cells and IFN-producing CD3+/CD8+ T cells (P=0.015, r=-0.62[-0.857, -0.15], P&amp;lt;0.01, r=-0.74[-0.913, -0.346], P=0.02, r=-0.6[-0.859, -0.107]). Intratumoral CD3+/CD4+ T cells did not significantly correlate with the rate of change in stiffness but did correlate with the rate of change in tumor size measured by B-mode ultrasound (P=0.03, r=-0.58[-0.850, -0.0730]). Conclusion: Our results confirm previous reports of immune escape in stiffer solid tumors in the context of HCC. Findings suggest that longitudinal monitoring of tumor stiffness can be useful for the assessment of immune cell infiltration. Citation Format: Kiyoyuki Minamiguchi, Andrea Cortes, Patricia Da Costa Lopez, Malea Williams, Simone Anfossi, Marites Melancon, Rony Avritscher. Longitudinal monitoring of stromal stiffness predicts immune escape in preclinical hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2396.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Quick and Easy Isolation of Immune Cells From Large-Volume Samples

Abstract Large-scale cell isolation is commonly performed in labs and core facilities for cell banking and drug discovery, and as a critical step in cell therapy manufacturing. Current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day for sample processing and cell isolation. To address this need, we have developed two methods: manual column-free isolation using the Easy 250 EasySep™ magnet, and automated cell isolation in a closed system using RoboSep™-C. The Easy 250 magnet allows users to isolate cells from a full leukopak of up to 20 billion cells in under 30 minutes by simply pipetting out their target cells. Negative selection protocols have been optimized to achieve 95.3% T cell, 97.2% CD4+ T cell, 91.9% CD8+ T cell, 99.4% B cell, 96.5% NK cell, and 91.5% monocyte purities. Positive selection protocols obtain 95.4% CD3+ cell, 94.4% CD4+ T cell, 93.9% CD8+ T cell, and 96.2% CD14+ cell purities. RoboSep™-C automates this cell isolation procedure, along with the cell washing steps for sample preparation, in as little as 50 minutes. The instrument features a scale tower, pump and clamp modules to direct fluid through defined paths, and a magnet for separation of labelled cells. The system uses a sterile single-use tubing set that incorporates a cartridge for cell washing and concentration, and a magnet chamber for cell separation. Starting with fresh leukopaks, we obtained 95.9% T cell, 95.8% CD4+ T cell, and 89.6% CD8+ T cell purities following negative selection, and 92.0% CD4+ T cell and 89.5% CD8+ T cell purities following positive selection. These approaches offer efficient and user-friendly cell isolation that allow researchers to scale up their operations, and can be easily integrated upstream of existing workflows.

Microfluidic cell sorting for gentle isolation of immune cells.

Abstract Isolation of specific immune cells is critical to biomedical research, however, techniques such as droplet-based fluorescence-activated cell sorting can mechanically irritate cells resulting in cell death or unwanted activation of immune cells that affect experimental analysis. Here we show that T cell subsets and neutrophils can be isolated using a low pressure (&amp;lt;2 psi) microfluidic cell sorter (WOLF G2®, NanoCellect®). These experiments show that this 11-parameter cell sorter can isolate complex cell types at high purity and high viability. In addition, due to the low-pressure microfluidic sorting mechanism, laminar flow reduces shear stress relative to traditional droplet sorters and cells remain functional and unaltered for subsequent experiments. Furthermore, we demonstrate high quality single-cell genomics data when combined downstream with single-cell partitioning and plate-based workflows. These findings demonstrate a novel workflow for isolating immune cells using the next generation of cell sorters.

Immune characterization of urothelial bladder carcinoma integrating transurethral resection of bladder tumor (TURBT) samples and serum: A feasibility study.

538 Background: Urothelial bladder cancer (UBC) is characterized by high immunogenicity. Multiple immunotherapies both in the perioperative and advanced setting are being developed. However, we lack good predictive biomarkers of response to immunotherapy in UBC. We aimed to characterize the immune profile of UBC patients using TURBT and serum samples. Methods: 13 UBC biopsies from TURBT samples were analyzed along with sera of these patients. Biopsy samples were divided in two equal parts. One part was formalin-fixed paraffin embedded and stained for immune histochemistry [IHC] for the following markers (CD3, CD4, CD8, CD20, CD68 and PD-L1 using SP142 antibody) and results expressed in percentages. The other part was homogenized and processed for immune cell isolation (human tumor isolation Miltenyi Kit). Tumor cells were stained with PHK and double positive CD8 and PHK were isolated using a MACSTM procedure. Different immune cell populations were analyzed by flow cytometry for the following markers (CD3, CD4, CD8, CD20 and CD68) and results expressed in percentages. Cytokines were examined in sera using the Luminex multiparametric procedure and results were expressed as pg/mL. Results: IHC and flow cytometry data were comparable and identified three groups according to the immune cells associated with the tumors (Table). Groups 1 and 2 showed high and moderate levels of tumor activated CD8+ T cells (1-4%) along with a higher expression of PD-L1 (40-20%). Group 3 lacked tumor activated CD8+ T cells and show lower expression of PD-L1 (15%). All groups showed significant levels of Th2 cytokines (IL-4, IL-6 or IL-10) while very low or lack of Th1 cytokines (IFN or IL-2) was observed except in group 2. Conclusions: TURBT biopsies and serum analysis of cytokines could represent a valid approach to further evaluate potential predictive biomarkers of response to immunotherapy in UBC. Different immune populations subgroups were identified in our series and could potentially predict for diverse responses to treatment. Further prospective validation in larger series is ongoing to confirm these results.[Table: see text]

Humanized Mouse Model to Study the P2X7 Receptor in Graft-Versus-Host Disease.

Humanized mouse models of graft-versus-host disease (GVHD), where human immune cells are injected into immune deficient mice, are well established and provide opportunities to investigate pathways involved in GVHD development. This chapter provides an overview of human immune cell isolation, injection of these cells into immune deficient mice, monitoring of mice for signs of GVHD, and assessment of human cell engraftment using flow cytometry. Further, this chapter focuses on the P2X7 signaling pathway involved in GVHD, and describes a strategy to block the P2X7 receptor and examine the effect of this on GVHD development.

Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro.

Simple SummaryKnowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, will allow the development of targeted nutrition strategies, prevent infectious diseases and the usage of antimicrobials, and promote the health of animals. To investigate the mechanisms of action of immunomodulating compounds, controlled in vitro systems using freshly isolated immune cells from blood represent a promising alternative to animal experiments. Immune cell isolation from the blood of chickens is a complex and difficult process since the immune cell fractions are significantly contaminated with red blood cells and platelets. To our knowledge, a robust protocol for immune cell isolation from chicken blood and the subsequent cultivation of immune cells is not available. Here, we established a protocol for blood sampling and immune cell isolation and cultivation from chicken blood, which could be applied for the investigation of direct effects of immunomodulating compounds. This protocol, combining different techniques of immune cell isolation, cultivation, and differentiation of distinct immune cell populations, will serve as a potential alternative to animal testing in vivo. By gaining knowledge about the mechanisms of action of immunomodulating compounds, this in vitro model will contribute to promote health and welfare in chicken farming.Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.

T Lymphocyte-Captured DNA Network for Localized Immunotherapy

The efficient isolation of immune cells with high purity and low cell damage is important for immunotherapy and remains highly challenging. We herein report a cell capture DNA network containing polyvalent multimodules for the specific isolation and in situ incubation of T lymphocytes (T-cells). Two ultralong DNA chains synthesized by an enzymatic amplification process were rationally designed to include functional multimodules as cell anchors and immune adjuvants. Mutually complementary sequences facilitated the formation of a DNA network and encapsulation of T-cells, as well as offering cutting sites of a restriction enzyme for the responsive release of T-cells and immune adjuvants. The purity of captured tumor-infiltrating T-cells reached 98%, and the viability of T-cells maintained ∼90%. The T-cells-containing DNA network was further administrated to a tumor lesion for localized immunotherapy. Our work provides a robust nanobiotechnology for efficient isolation of immune cells and other biological particles.

Combined Measurement of RNA and Protein Expression on a Single-Cell Level.

Single-cell RNA sequencing (sc-RNAseq) has become a critical approach for the analysis of immune cell function and heterogeneity. So far, the immune cell isolation, based on surface marker expression predicted by the RNA expression profiles, is often limited by the poor correlation between transcript and protein expression patterns. To overcome these difficulties, novel single-cell multi-omic approaches based on the combined analysis of transcript and surface protein expression have been developed. One of the major benefits of these technologies is the possibility to use a high number of antibodies conjugated with oligonucleotide (AbOs) for the surface marker detection, thus overcoming the limit of using few surface markers as occurs in flow cytometry. Here we describe the BD Rhapsody single-cell analysis system protocol for 3' mRNA whole transcriptome analysis (WTA), combined with AbO- and Sample Tag library preparation.