SEARCH · PubMed Health

Results for “Fluorescence-activated cell sorting (FACS)”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed2025

FACS-Proteomics strategy toward extracellular vesicles single-phenotype characterization in biological fluids: exploring the role of leukocyte-derived EVs in multiple sclerosis.

BACKGROUND: The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis. RESULTS: Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers. Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+ EVs), reaching a high level of purity (> 90%). Purified Leuko EVs were characterized using atomic force microscopy, nanoparticle tracking, and shotgun proteomics analysis revealing a similar coded protein cargo in both biological fluids. Subsequently, the same workflow was applied to tears from Relapsing-Remitting Multiple Sclerosis (RRMS) patients, revealing a Leuko EVs protein cargo enrichment that reflects the neuroinflammatory condition characteristics of RRMS. This enrichment was evidenced by the activation of upstream regulators TGFB1 and NFE2L2, which are associated with inflammatory responses. Additionally, the analysis identified markers indicative of endothelial cell proliferation and the development of enhanced vascular networks, with AGNPT2 and VEGF emerging as activated upstream regulators. These findings indicate the complex interplay between inflammation and angiogenesis in RRMS. CONCLUSIONS: In conclusion, our combined FACS-Proteomics strategy offers a promising approach for biomarker discovery, analysing cell-specific EV phenotypes directly from untouched body fluids, advancing the clinical value of tears EVs and improving the understanding of EV-mediated processes in vivo. Data are available via ProteomeXchange with the identifier PXD049036 and in EV-TRACK knowledgebase with ID: EV240150.

Humans↗

PubMed1992

Antibodies to the rat substance P receptor: production and characterization.

1. A protein A-rat substance P receptor (SPR) fusion protein was genetically engineered and used as an immunogen to raise a polyclonal antiserum to the SPR. The fusion protein was expressed in Escherichia coli driven by the heat-inducible lambda promoter (lambda Pr). 2. The fusion protein was purified using an IgG-Sepharose column, which specifically binds proteins containing the protein A moiety. The IgG fraction obtained after the immunization was cleaved to produce Fab fragments, which were subsequently purified using a fusion protein affinity column. The serum (anti-SPR Fab serum) was analyzed by fluorescence-activated cell sorting (FACS) and immunohistochemistry on both a constitutive cell line for the SPR (AR42J) and a cell line transfected with the SPR (KNRKSPR). 3. Specificity of the antiserum for SPR was confirmed by immunohistochemistry on cells using antiserum that had been preincubated with the protein A fusion protein (blocked). 4. The Ca2+ signal normally observed on stimulation of SPR with SP in AR42J cells and SP binding to KNRKSPR cells was shown to be diminished in the presence of anti-SPR Fab serum. SPR from both cell lines was immunoprecipitated using the anti-SPR Fab serum. The antiserum itself did not induce intracellular Ca2+ mobilization normally observed when cells were incubated with SP. 5. This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions.

Amino Acid Sequence↗

PubMed1992

Effect of enkephalins on bone marrow cells.

Mouse bone marrow cells were incubated with methionine- or leucine-enkephalin (10(-15)-10(-6) M) before seeding into soft agar cultures. In marrow samples harvested at different times, enkephalins decreased GM colony count on average by 30-40%. In individual experiments, however, the same concentration of enkephalins caused even stimulation, or at other times had effect. In view of the circadian periodicity of neuroendocrine functions and hematopoietic activity, the enkephalin effect on bone marrow cells was tested on marrow samples harvested at fixed time points (6 am, 6 pm), using enkephalin concentrations in the physiological range (10(-12)-10(-9) M). The seeding efficiency of the 6-pm cell population was on average 50% above that of the 6-am population. The 6-pm cell population was also more susceptible to the inhibitory effect of the enkephalins (35% inhibition) than the 6-am population (15% inhibition), and the variability in response was considerably reduced. With progenitor cell-enriched population, obtained by fluorescence-activated cell sorting (FACS) of 6-am bone marrow samples, in 3 out of 6 experiments Met- and Leu-enkephalin showed 30-35% inhibition of GM colony formation over a wide range of concentrations (10(-15)-10(-6)). In the other 3 experiments, suppression as well as stimulation or no alteration in colony count were observed. This variability probably reflected quality (purity) of the progenitor cell population, and may indicate that the enkephalins affected hematopoietic cells via a population of accessory cells.

Analysis of Variance↗

PubMed2026

Defining active and repressive chromatin states in neural crest cells using low-input CUT&RUN.

The transition of neural crest cells (NCCs) from a multipotent state to lineage-restricted derivatives, including melanocytes, is governed by tightly regulated epigenetic mechanisms that orchestrate cell type specific gene expression programs. Histone post-translational modifications (PTMs), in particular, play an important role in modulating chromatin accessibility, enhancer activation, and transcription factor occupancy, thereby facilitating dynamic chromatin and transcriptional reprogramming required during development. However, profiling such chromatin states in rare and transient Neural Crest Cell (NCC) populations in vivo remains technically challenging. To address this, we present an optimized low-input Cleavage Under Targets and Release Using Nuclease (CUT&RUN) workflow tailored for fluorescence-activated cell sorting (FACS) isolated NCCs from zebrafish embryos. This approach enables high-resolution and low-background mapping of key histone modifications, including H3K27ac, H3K4me3, and H3K27me3, from limited cell numbers. Collectively, these methodologies provide a robust framework for dissecting chromatin state dynamics in developmental systems and can also offer insights into epigenetic dysregulation associated with disease.

Animals↗

PubMed2026

Single-cell vector copy number analysis of phenotypically defined long-term hematopoietic stem cells for gene therapy safety assessment.

Hematopoietic stem cell (HSC)-based gene therapy has emerged as a transformative approach for the treatment of genetic diseases; however, accurate evaluation of vector copy number (VCN) remains critical for ensuring safety. Conventional bulk VCN assays, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR), do not resolve clonal heterogeneity and cannot identify rare high-VCN cells that may contribute disproportionately to insertional mutagenesis risk. Here, we developed an accessible single-cell VCN profiling method by combining fluorescence-activated cell sorting (FACS) of phenotypically defined long-term HSCs (Lineage- CD34+ CD38- CD90+ CD45RA- cells) with whole-genome amplification followed by conventional qPCR. This approach enabled resolution of VCN distributions at single-cell level using standard laboratory techniques. Notably, single-cell analysis revealed a high VCN tail that bulk VCN analysis could not resolve. Furthermore, in a humanized mouse transplantation model, single-cell VCN profiling demonstrated that overall VCN distributions could be analyzed after engraftment, although inter-donor and inter-mouse variability was observed. Collectively, this method provides a rapid, cost-effective, and phenotypically resolved strategy for assessing VCN heterogeneity in gene-modified HSCs. Single-cell VCN profiling offers complementary insights beyond conventional bulk assays and may enhance preclinical safety evaluation of gene and cell therapy products.

lentiviral vector↗

PubMed2025

Live-Cell Monitoring and Omics Analysis of Liquid-Solid Transitions of Biomolecular Condensates.

Biomolecular condensates, or so-called membraneless organelles, transition from liquid into more solid-like states over time, contributing to the development of pathological conditions. The present study proposes a simple method using photoactive yellow protein (PYP) and its specific fluorescent covalent ligands to distinguish between the liquid and solid states of protein condensates in live cells. The method, compatible with fluorescence-activated cell sorting (FACS), correlates the stiffness of specific protein condensates with their accessibility to PYP ligands, enabling quantitative multicolor monitoring of condensate solidification. We applied this technique to 12 phase-separating proteins and their mutants, finding that TDP-43, particularly its A315T mutant linked to familial amyotrophic lateral sclerosis, most readily forms solid aggregates. Furthermore, this FACS-compatible strategy enabled the isolation of distinct cell populations based on condensate states, allowing for subsequent proteomic and transcriptomic analyses. Our findings demonstrate that condensate solidification is accompanied by the upregulated expression of extracellular matrix proteins, suggesting a previously unrecognized link between solid aggregate formation and extracellular matrix hardening.

Humans↗

PubMed2026

VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature.

The human cerebrovasculature is a critical yet historically understudied component of neurological health. Dysfunction of the diverse endothelial, mural, and perivascular cells that comprise cerebral vessels is central to diseases ranging from stroke to Alzheimer's disease. However, characterizing these cell populations at a molecular level has proven exceptionally challenging. Encased within a robust basement membrane, vascular cells resist standard dissociation methods, leading to their systematic depletion and underrepresentation in existing single-nucleus genomic atlases. This has created a major blind spot in neuroscience. To overcome this barrier, we developed vessel isolation and nucleus extraction for sequencing (VINE-seq) and its advanced iteration, MultiVINE-seq. The protocol provides a robust, reproducible workflow for the enrichment and high-resolution profiling of vascular, perivascular, and immune cells from fresh or frozen human and mouse brain tissue. First, intact vessels (predominantly capillaries and small arterioles/venules, 100 µm in diameter) are isolated from homogenized brain tissue via dextran-based density-gradient centrifugation, separating the vascular pellet from myelin and the parenchymal fraction. Second, the collected vessels are rigorously washed over a cell strainer to remove trapped contaminants. A critical innovation lies in the third stage: the optimized extraction of nuclei from purified vessels using enzymatic digestion. After extraction, the protocol uses fluorescence-activated cell sorting (FACS) to ensure collection of high-purity nuclei suitable for widely used droplet-based sequencing platforms (e.g., 10x Genomics single cell 3' or multiome). This protocol requires 4-5 h to complete and can be carried out by researchers with single-cell and flow cytometry training.

Journal Article↗

PubMed1992

Alterations of lymphocyte populations during development in the spontaneously hypertensive rat.

OBJECTIVE: Immune system abnormalities have been linked to hypertension in the spontaneously hypertensive rat (SHR). The goal of our study was to examine different lymphocyte subpopulations in the prehypertensive and developmental phases of hypertension in the SHR. DESIGN: Blood samples were obtained from SHR and Wistar-Kyoto (WKY) rats at the following time-points: 2 weeks and 1, 2, 3 and 4 months. Lymphocytes were separated from the whole blood. METHODS: Monoclonal antibodies were used to fluorescently label the following lymphocyte subpopulations; total T cells, T non-helper cells, T helper cells and B cells. Fluorescence-activated cell sorting (FACS) analysis was used to quantify the percentages of the different subpopulations examined. RESULTS: The T non-helper cell population was depressed in SHR from 2 weeks of age. This finding persisted throughout the entire 4-month study period. At the 4-month time-point, the total T cell percentage was also depressed in the SHR. CONCLUSIONS: These results demonstrate that immune system abnormalities are present in the prehypertensive and developmental phases of hypertension in the SHR. This supports the hypothesis that the immune system is involved in the development and maintenance of hypertension in the SHR, preceding not adapting to this state.

Animals↗

PubMed1992

Clonal involvement of granulocytes and monocytes, but not of T and B lymphocytes and natural killer cells in patients with myelodysplasia: analysis by X-linked restriction fragment length polymorphisms and polymerase chain reaction of the phosphoglycerate kinase gene.

To determine the clonal nature of hematopoiesis and to assess lineage involvement in patients with myelodysplastic syndromes (MDS), we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Eleven female MDS patients heterozygous for at least one of these probes were studied: 3 with refractory anemia (RA), 2 with RA with ringed sideroblasts (RARS), 2 with chronic myelomonocytic leukemia (CMML), and 4 with RA with excess of blasts in transformation (RAEB-t). All exhibited clonal hematopoiesis as determined by Southern analysis of DNA prepared from peripheral blood (PB) and/or bone marrow (BM) cells. In three of the six patients heterozygous for the PGK1 gene, purified cell suspensions of polymorphonuclear cells (PMN), monocytes, lymphocytes, and/or T cells prepared from PB were tested. In addition, five of these patients were analyzed by a polymerase chain reaction (PCR)-based procedure as described recently. This method was slightly adapted to facilitate the analysis of cell lysates of fluorescence-activated cell sorted (FACS) monocytes, T and B lymphocytes, and natural killer (NK) cells. The outcome of Southern and PCR analysis was concordant, showing that PMN and monocytes were clonally derived, whereas circulating T and B lymphocytes and NK cells exhibited random X-chromosome inactivation compatible with a polyclonal pattern. To address the question of whether T cells are derived from unaffected progenitor cells or that their origin had antedated the onset of MDS, naive and memory T cells were analyzed separately. Both subsets showed a polyclonal pattern. However, in one patient analysis of constitutive DNA suggested a skewed methylation, and the presence of clonal lymphocytes against a background of polyclonal lymphoid cells cannot be ruled out in this patient. PCR analysis of PB and BM cells showed a nonrandom, unilateral pattern of X-inactivation, compatible with a mixture of clonally (myeloid) and polyclonally (lymphoid) derived cells. In conclusion, in some patients, MDS represents a disorder with clonal hematopoiesis restricted to cells of myeloid origin, whereas a random X-inactivation pattern is found in lymphoid cells.

Adult↗

PubMed1992

Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system.

A gramicidin S (GS) hyperproducing mutant of Bacillus brevis was isolated by using a protein-staining fluorescence dye (fluorescein isothiocyanate, FITC), and a fluorescence-activated cell sorting system (FACS). By flow cytometry (FCM) analysis after staining with FITC, higher producing cells of the wild-type had higher fluorescence signals than cells with low productivity or cells from a GS non-producing mutant. Staining with FITC did not affect the viability of cells under the conditions chosen for FCM analysis. This enabled us to recover viable cells after sorting. After wild-type cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, mutants with higher fluorescence than the parental strain were obtained by cell sorting. Among them, strain 18 was chosen as a GS hyperproducer; it produced 590 micrograms GS/ml compared to 350 micrograms/ml by the wild-type strain. This method has the advantage of being able to screen large numbers of cells in a short time. Furthermore, use of the fluorescence dye technique will expand the use of FACS to the improvement of other cultures that produce metabolites that do not have a specific fluorescence or strong enough fluorescence for normal cell sorting.

Bacillus↗

PubMed2025

Genome editing with programmable base editors in human cells.

Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.

Humans↗

PubMed1978

The relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. III. Expression of a single predominant isotype on primed and unprimed B cells.

We determined whether primed and unprimed B cells in the spleen of (BALB/c x C57BL/Ka)F(1) mice contain subpopulations that express a predominant surface Ig isotype. Spleen cells were stained for surface isotypes and sorted on the fluorescence-activated cell sorter (FACS) in order to obtain B cells bearing predominantly IgM (mup cells), IgD (deltap cells), or IgG (gammap cells). Each population was assayed for its capacity to restore the adoptive primary and secondary anti-bovine serum albumin (BSA) antibody response in irradiated syngeneic recipients. In addition, the adoptive response restored by isotype-predominant cells was compared to that restored by isotype- positive cells (B cells bearing a given surface isotype alone or in combination with others). The experimental results show that mup cells restore the adoptive primary and secondary IgM and IgG responses to BSA, and gammaP cells restore only the primary and secondary IgG response. Deltap Cells restored the adoptive secondary IgG response, but failed to restore the adoptive primary response at the cell doses tested. GammaP Cells but not deltap cells suppressed the IgM response of the mu(+) and delta(+) cells. The contribution of isotype-predominant cells to both the adoptive primary and secondary anti-BSA response was smaller than that of B cells bearing a combination of surface isotypes. Differences in the Ig isotype pattern expressed on the surface of primed and unprimed B cells are discussed.

Animals↗

PubMed1979

An improved method for selecting T-cell hybridomas by the fluorescence-activated cell sorter.

A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described to select T cell hybridomas from cell populations obtained from the fusion of activated T cells and T- lymphoma cells. Cells are first sorted on the basis of differences in cell-surface antigens. After a period of growth in culture, the cells are then stained with the DNA-labeling dye dis-benzimidazole 33258 Hoechst and sorted on the basis of their DNA content. From 15 to 25% of the resulting cell clones, growing out of limiting dilutions, were hybrid lines as judged by chromosome analysis.

Animals↗

PubMed1979

Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter.

Myeloma-spleen cell hybrids (hybridomas) producing antibody to mouse immunoglobulin allotypes have been labeled with fluorescent microspheres coupled with myeloma protein antigens. The ratio of specific to nonspecific microsphere binding by viable hybridoma cells was about 100:1. By using a modified fluorescence-activated cell sorter (FACS), selected hybridoma cells in a mixture have been sorted individually into media in microculture wells, where, with thymocyte feeder cells, they developed into clones producing a desired monoclonal antibody. Viable cells were selected by measurement of their light scattering and autofluorescence properties. Rare antibody-producing clones were obtained without laborious screening and repeated subculturing. This technique should expand the range of monoclonal antibodies readily obtained from hybridomas and greatly facilitate the process of obtaining desired hybridomas.

Animals↗

PubMed1992

Synergistic effect of IL-3 and IL-6 on highly enriched murine hemopoietic progenitors.

It has been reported that interleukin 6 (IL-6) acts on hemopoietic stem cells in synergism with interleukin 3 (IL-3), but it has not yet been clarified whether IL-6 acts directly on the stem cells or not. To investigate the mechanism of the synergism between IL-3 and IL-6, we sorted hemopoietic stem cells from untreated murine bone marrow cells using a two-laser fluorescence-activated cell sorter (FACS). Cells negative for the lymphohemopoietic lineage (lineage-negative, Lin-), with a high affinity to wheat germ agglutinin (WGA+), and showing a low expression of Thy-1 antigen (Thy-1low) were sorted and analyzed by in vitro and in vivo colony formation. This fraction was 0.4% of the total mononuclear bone marrow cells. Approximately 25% of these Lin-WGA+Thy-1low cells showed in vitro colony formation, whereas approximately 1% of them formed day-8 and day-12 spleen colonies. Thus, it appears that the Lin-WGA+Thy-1low cells were a highly enriched stem cell population. By FACS clone sorting, single cells were isolated from the enriched stem cell fraction and cultured in semisolid or liquid culture systems. Addition of IL-6 to methylcellulose medium containing IL-3 did not significantly increase the number of colonies. It is thus suggested that the target cells of IL-3 and IL-6 are the same as those of IL-3. The secondary colony-forming ability of primary colonies that developed in the presence of IL-6 and IL-3 was higher than that of colonies formed in the presence of IL-3 alone. In correspondence with this finding, the numbers of myeloid colonies and spleen colony-forming units (CFU-S) were increased by the incubation of these sorted cells for 7 days with IL-6 and IL-3 when compared with the effect of IL-3 alone. Therefore, it is concluded that IL-6 acts directly on hemopoietic stem cells to enhance their proliferation.

Animals↗

PubMed1979

Maturation of B lymphocytes. II. Sequential appearance of increasing IgM and IgD in the adult bone marrow.

The relationship between surface IgM (sIgM) and surface IgD (sIgD) was examined on small lymphocytes in the adult murine bone marrow or prepubertal spleen. Cells were sorted on the basis of different sIgM levels by a fluorescence-activated cell sorter (FACS) and relabeled for sIgD or total sIg by a sandwich technique using 125I-labeled protein A and radioautography. For detecting sIgD, an anti-delta allotype reagent was used in congenic mice. Cells lacking sIgM in the bone marrow or spleen were also found to be sIgD-; thus, sIgD appeared only in the presence of sIgM. Weak sIgM-bearing cells in the bone marrow also had no sIgD indicating that sIgD appeared only after the acquisition of a significant level of sIgM. Subsequently, the incidence of sIgD+ cells increased in fractions showing increasing sIgM levels indicating the acquisition of new sIgD by "sIgM only" cells with increasing maturation levels in the bone marrow. In marrow lymphoid cells expressing both Ig isotypes, sIgM and sIgD levels increased in parallel, possibly with increasing maturation level. In the spleen, the incidence of sIgD+ cells among various cell fractions showing different sIgM levels was found constant. However, spleen cells bearing both receptors, showed a small increase in the sIgD level with increasing sIgM level.

Aging↗

PubMed1978

Two functionally distinct subpopulations of human T cells that collaborate in the generation of cytotoxic cells responsible for cell-mediated lympholysis.

A human thymus-dependent differentiation antigen, TH2 was defined by a rabbit anti-human T cell serum absorbed with autologous B lymphoblasts and leukemic cells bearing T cell markers from a patient with chronic lymphocytic leukemia. Anti-TH2 reacted specifically with thymus-derived lymphoid cells and exhibited two distinct profiles of reactivity with normal peripheral T cells as detected by indirect immunofluorescence on a FACS I. Isolation of strongly reactive, TH2+, from weakly reactive, TH2- T cells by fluorescence-activated cell sorting revealed that the TH2+ subset contained most of the killer activity in cell-mediated lympholysis (CML), but had a diminished response in MLC and a suboptimal or negligible proliferative response to soluble antigens (mumps, PPD, tetanus toxoid). In contrast, the TH2- subset contained markedly less killer activity but amplified cytotoxicity by TH2+ cells and exhibited a proliferative response to both alloantigen and soluble antigens that was often significantly greater than the response by unseparated T cells. The relevance of these findings to previously described human T cell subsets and to functional subpopulations of murine T cells is discussed.

Absorption↗

PubMed2024

Revealing differential expression patterns of piRNA in FACS blood cells of SARS-CoV-2 infected patients.

Non-coding RNA expression has shown to have cell type-specificity. The regulatory characteristics of these molecules are impacted by changes in their expression levels. We performed next-generation sequencing and examined small RNA-seq data obtained from 6 different types of blood cells separated by fluorescence-activated cell sorting of severe COVID-19 patients and healthy control donors. In addition to examining the behavior of piRNA in the blood cells of severe SARS-CoV-2 infected patients, our aim was to present a distinct piRNA differential expression portrait for each separate cell type. We observed that depending on the type of cell, different sorted control cells (erythrocytes, monocytes, lymphocytes, eosinophils, basophils, and neutrophils) have altering piRNA expression patterns. After analyzing the expression of piRNAs in each set of sorted cells from patients with severe COVID-19, we observed 3 significantly elevated piRNAs - piR-33,123, piR-34,765, piR-43,768 and 9 downregulated piRNAs in erythrocytes. In lymphocytes, all 19 piRNAs were upregulated. Monocytes were presented with a larger amount of statistically significant piRNA, 5 upregulated (piR-49039 piR-31623, piR-37213, piR-44721, piR-44720) and 35 downregulated. It has been previously shown that piR-31,623 has been associated with respiratory syncytial virus infection, and taking in account the major role of piRNA in transposon silencing, we presume that the differential expression patterns which we observed could be a signal of indirect antiviral activity or a specific antiviral cell state. Additionally, in lymphocytes, all 19 piRNAs were upregulated.

Humans↗