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PubMed1985

Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells.

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.

Cell Cycle↗

PubMed1997

Molecular single-cell analysis reveals that CD5-positive peripheral blood B cells in healthy humans are characterized by rearranged Vkappa genes lacking somatic mutation.

B cells expressing the CD5 cell surface antigen are involved in certain B cell malignancies and autoimmune diseases. From studies in the mouse, it emerged that CD5+ B cells represent a separate lineage of B lymphocytes that, in contrast to conventional (CD5-) B cells, are not driven into T cell-dependent immune responses in which rearranged variable (V) region genes are diversified by somatic hypermutation. Against this background it came as a surprise that human disease-involved CD5-positive autoreactive B cells as well as B cell chronic lymphocytic leukemias can harbor somatically mutated V region genes. Recent V gene analyses on CD5+ B cells in healthy adults did not give rise to a clear picture about the fraction of somatically mutated among all CD5+ B cells. In this work we used a molecular single-cell analysis to determine reliably the frequency of mutated CD5+ B cells in healthy humans: single, kappa light chain-expressing CD5+ peripheral blood B cells were isolated by flow cytometry, and rearranged Vkappa genes were amplified by PCR. From one donor, CD5+CD19+ B cells were analyzed. Since CD5+ B cells were found among IgM+IgD+ and IgM+IgD- cells (but almost not among class-switched cells) from two other donors, individual cells corresponding to these IgM-expressing subsets were investigated separately. The sequence analysis of rearranged Vkappa genes revealed that most if not all CD5+ B cells in healthy humans carry unmutated V region genes. From one of the donors, a novel polymorphic Jkappa2 gene segment was identified. To explain the discrepancy between the frequent occurrence of disease-associated somatically mutated CD5+ B cells and the low incidence or absence of somatic mutation in normal CD5+ B cells, we speculate that CD5+ B cells usually do not participate in germinal center reactions, but if they occasionally do so, they may be at an increased risk to become involved in autoimmune diseases or B cell malignancies.

Adult↗

PubMed1998

Single-cell analysis of lymphokine imbalance in asymptomatic HIV-1 infection: evidence for a major alteration within the CD8+ T cell subset.

In this study we investigated at single-cell level by flow cytometry the potential of T cell cytokine production in asymptomatic HIV-1-infected subjects with > 200 CD4 counts and possible correlation with T helper cell depletion and viral load. Mitogen-stimulated peripheral blood mononuclear cells from 32 HIV-1+ patients and 16 healthy subjects were intracytoplasmically stained for IL-2, interferon-gamma (IFN-gamma), IL-4 or IL-10, and the frequency of cytokine-producing cells was assessed in total T cells, CD4, CD8 and CD45RO subsets as well as in CD69+CD3+ gated lymphocytes. HIV-1+ patients, irrespective of their degree of CD4 depletion, exhibited a major increase in IFN-gamma+ CD8 T cells, largely due to CD28- cells, as well as a decrease in the capacity of CD8 T cells to produce IL-2. Patients with > 500 CD4 counts showed a diminished frequency of IL-4 expression in CD4 T cells and a negative correlation was found between this parameter and the ex vivo CD4 counts in the 32 patients. Analysis of patients stratified according to viral load revealed a significantly higher proportion of IL-2-producing CD4 cells in the group with < 5000 RNA copies/ml. In short, using single-cell analysis and an antigen-presenting cell-independent stimulus, we have not been able to find any significant cytokine imbalances in the CD4 subset, suggesting that the well described T helper defects are not due to intrinsic alterations in the potential of CD4 T cells to produce cytokines. On the other hand, the major disturbances in the CD8 T lymphocytes agree with the marked activation and possible replicative senescence of CD8 T cells and emphasize the role of this subset in HIV immunopathogenesis.

Adult↗

PubMed1998

Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis.

OBJECTIVE: To determine the cytokine profile of CD4+ T cells in the synovial tissue (ST) of rheumatoid arthritis (RA) patients at the single-cell level. METHODS: Unseparated ST cells and paired CD4+ T cells separated from the peripheral blood (PB) and ST of RA patients were stimulated for 4 hours with phorbol myristate acetate (PMA) plus calcium ionophore A23187, or for 6 hours with immobilized anti-CD3 plus anti-CD28, in the presence of brefeldin A. Cells were stained for intracellular cytokines such as interferon-gamma (IFNgamma), interleukin-2 (IL-2), IL-4, IL-10, and IL-13, in combination with cell surface markers. The percentages of cytokine-producing T cells were analyzed by flow cytometry. RESULTS: When ST cells were stimulated with PMA plus A23187 in bulk culture, IFNgamma-producing T cells were more frequently detected in the CD8+ subset, but cells producing other cytokines were found in the CD4+ subset. Purified ST CD4+ T cells, after stimulation with PMA plus A23187, were able to produce higher levels of IFNgamma but lower levels of IL-4 and IL-13, by analysis at the single-cell level, as compared with the PB CD4+, CD45RO+ T cells. The majority of IL-4- or IL-13-producing ST CD4+ cells produced IFNgamma, although PB CD4+ T cells rarely showed this cytokine pattern. IL-10-producing CD4+ T cells were more frequently found in the ST than in the PB. Of interest, most of the IL-10-producing ST CD4+ T cells were able to produce IFNgamma. IL-2-producing CD4+ T cells were similarly present in both compartments. Similar intracellular cytokine patterns were observed with anti-CD3 plus anti-CD28 stimulation, although the number of detected cells was lower. CONCLUSION: These data indicate that CD4+ T cells present within the inflamed synovium have apparently distinct cytokine profiles from those of memory CD4+ T cells in the PB, as typified by their ability to secrete both IFNgamma and IL-10.

Aged↗

PubMed2004

Microtechnologies and nanotechnologies for single-cell analysis.

Many efforts are currently underway to try and mimic the properties of single cells with the aim of designing chips that are as efficient as cells. However, cells are nature's nanotechnology engineering at the scale of atoms and molecules, and it might be better to envision a microchip that utilizes a single cell as an experimentation platform. A novel, so-called laboratory-in-a-cell concept has been described, where advantage is taken of micro- and nanotechnological tools to enable precise control of the biochemical cellular environment; these tools also offer the possibility to analyse the composition of single cells. Methods for single-cell handling and analysis are being developed and will be required for this concept to progress further.

Animals↗

PubMed2000

Single-cell analysis of bacteria by Raman microscopy: spectral information on the chemical composition of cells and on the heterogeneity in a culture.

In the acetone-butanol (ABE) fermentation process, the utilised organisms from the group of the solventogenic Clostridia go through a complex cell-cycle. The role of different cell types in product formation is not understood in detail yet. We aim to use Raman spectroscopy to characterise the population distribution in Clostridium cultures. Cell suspensions were dried on calcium fluoride carriers. Raman spectra of single cells were obtained using a confocal Raman microscope (Dilor, Lille, France). The laser beam was focused on individual cells through the microscope objective. Spectra with good signal-to-noise ratio were obtained. Cells of different morphology, but also apparently similar cells, showed different spectra. Several cell components could be detected and varied in quantity. Compared to other methods for single-cell analysis, the new method is much more time-consuming to analyse one individual cell. However, a large amount of chemical information is obtained from each single cell in a non-destructive, non-invasive way. Raman microscopy appears to be a suitable method for studying population distributions in bacterial cultures.

Clostridium↗

PubMed2000

Single cell analysis of cytokine expression kinetics by human CD4+ T-cell clones during activation or tolerance induction.

Exposure to optimal peptide antigen concentrations induces human CD4+ T-cell clones to proliferate and secrete various cytokines. Higher (> 10-fold optimal) antigen concentrations cause long-term proliferative unresponsiveness, which can be reversed by exogenous interleukin-2 (IL-2). We call this condition 'tolerance'. We used intracellular cytokine staining and flow cytometric analysis to investigate the kinetics of interferon-gamma, tumour necrosis factor-alpha, IL-4 and IL-5 production during the initial phase of tolerance induction. Single cell analysis of interferon-gamma and IL-4 or IL-5 coexpression showed functional heterogeneity of cloned human CD4+ T cells. Superstimulation with phorbol 12-myristate 13-acetate and ionomycin (PI) revealed enhanced responsiveness shortly after tolerizing treatment, followed by reduced responsiveness. Both tolerized and activated T cells had similarly reduced cytokine responses when further stimulated with antigen during the following 48 hr, with limited enhancement following additional stimulation with PI. We conclude that cytokine induction is normally followed by a refractory phase, but that the expression of cytokines is enhanced in the initial phase of tolerance induction.

Antigens↗

PubMed2005

Cytomics, the human cytome project and systems biology: top-down resolution of the molecular biocomplexity of organisms by single cell analysis.

A large amount of structural and functional information is obtained by molecular cell phenotype analysis of tissues, organs and organisms at the single cell level by image or flow cytometry in combination with bioinformatic knowledge extraction (cytomics) concerning nuclei acids, proteins and metabolites (cellular genomics, proteomics and metabolomics) as well as cell function parameters like intracellular pH, transmembrane potentials or ion gradients. In addition, differential molecular cell phenotypes between diseased and healthy cells provide molecular data patterns for (i) predictive medicine by cytomics or for (ii) drug discovery purposes using reverse engineering of the data patterns by biomedical cell systems biology. Molecular pathways can be explored in this way including the detection of suitable target molecules, without detailed a priori knowledge of specific disease mechanisms. This is useful during the analysis of complex diseases such as infections, allergies, rheumatoid diseases, diabetes or malignancies. The top-down approach reaching from single cell heterogeneity in cell systems and tissues down to the molecular level seems suitable for a human cytome project to systematically explore the molecular biocomplexity of human organisms. The analysis of already existing data from scientific studies or routine diagnostic procedures will be of immediate value in clinical medicine, for example as personalized therapy by cytomics.

Cell Biology↗

PubMed2026

Single-cell analysis of the human retina reveals stage-linked microglial states and neural-immune circuit rewiring in diabetic retinopathy.

Diabetic retinopathy (DR) is a major cause of vision loss worldwide. Here, we conduct single-cell RNA sequencing of twenty human retina samples (from living and post-mortem donors) across non-diabetic, diabetic, and DR states to create a comprehensive transcriptomic atlas. We identify two stable microglial populations-homeostatic and inflammatory-that exist along a functional continuum, plus a neutrophil cluster within C1QA+ myeloid cells with dynamic transitions occurring throughout disease progression. Module-level analysis reveals divergent transcriptional trajectories: homeostatic microglia maintain energetic programs while selectively upregulating stress elements, whereas inflammatory microglia layer additional pro-inflammatory programs onto preserved biosynthetic foundations. Eleven co-expression modules organize into two major axes: an inflammatory-stress axis, and a regulatory/metabolic-motility axis, with a stable translation module persisting across disease stages. Cell communication analysis further highlights sophisticated neural-immune interactions, particularly between photoreceptors and microglia. Our findings provide insights into the complex cellular dynamics of DR progression and suggest potential therapeutic targets for early intervention.

Humans↗

PubMed1993

Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations.

We have used a single-cell based polymerase chain reaction (PCR) amplification technique to examine the gene expression pattern in single Hodgkin's and Reed-Sternberg (H&RS) cells from seven patients with Hodgkin's disease. Single cells were isolated from lymph nodes obtained at diagnosis (5 of 7 patients) or in first or second relapse (2 of 7 patients). Gene expression was examined by hybridization to a panel of 22 cDNA probes. Forty-nine H&RS cells (and 23 CD3+ or CD20+ lymphocytes as controls) from four patients with nodular sclerosing Hodgkin's disease (HD) and one patient each with lymphocyte predominant and mixed-cellularity HD were successfully analyzed by PCR. This analysis provides evidence that single H&RS cells can coexpress genes characteristic of several hematopoietic lineages (monocytes and lymphocytes). Genes characteristic of activated lymphoid cells are expressed in most H&RS cells. Heterogeneity of expression for certain genes between different cases was found and may eventually define molecular subgroups of HD. These findings indicate that H&RS cells of HD resemble activated hematopoietic cells. Phenotypically similar cells from different cases exhibit characteristic molecular differences. In one patient, 5 of 7 single RS cells showed identical p53 cDNA mutations at codon 246 on specific reverse transcriptase [RT]-PCR and sequencing of exons 5 through 8. The novel experimental approach may provide a valuable tool for understanding the molecular events in newly diagnosed Hodgkin's disease and progression of the disease.

Adolescent↗

PubMed2000

A single cell analysis of apoptosis. Ordering the apoptotic phenotype.

Microcinematography was applied to the analysis of the kinetics of apoptotic cell death. Apoptosis was found to be a process that proceeds in different cells at different times after an initial stress, and therefore kinetic studies of apoptotic events in bulk cultures can be problematic. Using single cell analysis we found that stronger apoptotic stimuli induce an earlier onset of apoptosis, but that there is no relationship between time of onset and duration of the apoptotic process. That is, cells that initiate apoptosis shortly after induction do not proceed more rapidly through the process. This suggests an all-or-non-mechanism that is supported by some models of the biochemical pathways of apoptosis.

Apoptosis↗

PubMed2026

A scalable, low-cost, sample hashing workflow for multiomic single-cell analysis using the Seq-Well S3 platform.

In-depth analyses of clinical samples have the potential to provide unparalleled insights into the cellular mechanisms that underlie both health and disease, as well as therapeutic and prophylactic responses. However, these specimens are often paucicellular, necessitating the use of workflows that maximize the amount of information that can be learned. Here we provide a detailed protocol for generating and analyzing single-cell multiomic data from low-input samples with the Seq-Well S3 platform. We further describe a matched pipeline for sample hashing that reduces costs and sources of technical variation in the resulting data while also enhancing throughput. In brief, our streamlined and efficient methodology involves: (1) optionally staining single-cell suspensions with antibody-oligonucleotide conjugates for cell surface protein quantification and/or sample multiplexing; (2) generating Seq-Well S3 sequencing libraries; (3) optionally producing bulk-RNA sequencing libraries via SMART-seq2 to support genetic demultiplexing; and (4) computationally analyzing the resulting data. Each step herein has been designed to leverage readily available reagents and standard laboratory equipment, substantially lowering barriers to entry for researchers. The overall Protocol can yield high-quality multiomic insights from samples in under a week.

Single-Cell Analysis↗

PubMed1996

Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis.

BACKGROUND: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected embryos following in vitro fertilisation (IVF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation. METHODS AND RESULTS: Using nested PCR, the delta F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the delta F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify, apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from delta F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification. CONCLUSIONS: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.

Alleles↗

PubMed2026

Identification of Drug-resistant Cell Subpopulations in Colorectal Cancer Through Single-cell Analysis and Exploration of Potential Therapeutic Strategies.

INTRODUCTION: The therapeutic efficacy of Colorectal Cancer (CRC) is often compromised by resistance to the standard chemotherapy agent oxaliplatin. METHODS: This study obtained single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database. Differentially Expressed Genes (DEGs) between resistant and sensitive epithelial subpopulations were identified, followed by enrichment analysis. Pseudotemporal trajectory and cell-cell communication were analyzed using Monocle2 and CellChat, respectively. The candidate drug was predicted by Connectivity Map (cMAP) analysis. External validation included assessment of the EpC2 signature in an oxaliplatin-resistant cell line dataset (GSE76092), survival analysis using The Cancer Genome Atlas (TCGA) cohorts, and re-analysis of the GSE179784 dataset to assess the reproducibility of EpC2-like subpopulations and their DNA Damage Repair (DDR) scores. RESULTS: Cell subpopulations were divided into 10 clusters. Among them, epithelial cells comprised 5 subpopulations, with EPC2 identified as a potential oxaliplatin-resistant subset. DEGs were enriched in the TNF and IL-17 pathways. External validation confirmed the enrichment of EpC2 in resistant cell lines and its association with poor survival. Pseudotemporal trajectory revealed that epithelial cells underwent state transitions, forming two distinct branches. The resistant group exhibited enrichment in RNA splicing and NF-κB pathways. Cell-cell communication analysis revealed interactions involving MDK- NCL and PPIA-BSG. Dasatinib was predicted as a candidate drug. DISCUSSION: We identified an oxaliplatin-resistant subpopulation of Epithelial Cells (EpC2) in CRC, elucidated its multi-layered resistance mechanisms, and integrated multi- omics and cMAP database analyses to predict a potential intervention drug. CONCLUSION: This study provided potential therapeutic possibilities for oxaliplatin resistance, contributing to CRC treatment.

Humans↗

PubMed1989

Single-cell analysis of Ca++ changes in human lung mast cells: graded vs. all-or-nothing elevations after IgE-mediated stimulation.

Human lung mast cells were examined by digital video microscopy for changes in cytosolic free ionized calcium [( Ca++]i) after stimulation with anti-IgE antibody or specific antigens. These studies sought to determine whether the mast cell response resembled a graded or an all-or-nothing process. Preliminary experiments indicated that labeling mast cells with fura-2 did not alter their response to IgE-mediated stimulation. Subsequent experiments established that an IgE-mediated stimulus evoked an elevation of [Ca++]i from a baseline value of 85 nM to an average of 190 nM (range 60-450 nM, n = 23), with an average histamine release of 26%. There was a good correlation (Rs = 0.67) between the average net [Ca++]i change and the subsequent histamine release (regression equation: %HR = 0.189[net(Ca)-52]). [Ca++]i elevations were found to precede histamine release (t1/2 for [Ca++]i of 35 s vs. t1/2 for histamine release of 110 s). Single-cell analysis found that even for very low values of histamine release, nearly all cells demonstrated a [Ca++]i response. However, this response was markedly heterogeneous, ranging from no response to responses two to three times the mean. Comparative studies of mast cells stimulated under optimal and suboptimal conditions established that there was a graded [Ca++]i response dependent on the strength of the stimulus. An all-or-nothing reaction for the [Ca++]i response was ruled out.

Antigens, Differentiation, B-Lymphocyte↗

PubMed2004

Apoptosis induced kinetic changes in autofluorescence of cultured HL60 cells-possible application for single cell analysis on chip.

INTRODUCTION: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. METHODS: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-alpha in combination with cycloheximide (CHX), or irradiated with 6 or 10 Gray, during varying time periods, to initiate apoptosis. AF was measured at the flow cytometer. RESULTS: Induction of apoptosis results in the shrinkage of the cell and the fragmentation into apoptotic bodies. With flow cytometry, 4 subpopulations, viable, early apoptotic, late apoptotic and the necrotic cells, can be distinguished. Induction of apoptosis results in a decrease in AF intensity compared to untreated HL60 cells, especially seen in the late apoptotic subpopulation. The AF intensity is found to decrease significantly in time (between 2 h and 24 h) for all the four apoptotic inducers used. CONCLUSIONS: Our results show that it is possible to specifically measure the apoptotic-induced kinetic changes in AF in HL60 cells. A decrease in AF intensity is seen from 2 h till 24 h. These results open a door for future developments in single-cell analysis.

Apoptosis↗

PubMed2001

Evaluation of immune activation in HIV-infected and uninfected African individuals by single-cell analysis of cytokine production.

Immune activation has been observed in HIV-infected and uninfected Africans, among whom it is thought to modify interaction between the immune system and HIV. To characterize this phenomenon accurately, in-depth immunologic analyses were performed in a rural African population. Freshly drawn peripheral blood mononuclear cells (PBMCs) of HIV-infected African (from Gulu, Uganda) and Italian antiviral-naive patients and those of uninfected Ugandan and Italian study subjects were analyzed. Individuals were matched for age and sex and determined to be free from parasitic infections. Intracellular cytokines were measured in mitogen (M)- and gp160 peptides + staphylococcal enterotoxin B and alpha CD28 (env)-stimulated T lymphocytes. Interferon (IFN)-gamma-producing CD8(+) T cells were quantified in an enzyme-linked immunosorbent assay. Results showed that M-stimulated production of interleukin (IL)-10 and tumor necrosis factor (TNF)-alpha increases in CD4(+) and CD8(+) cells of African infected patients and uninfected study subject; and that env-stimulated IL-10 and TNF-alpha production is increased in CD8(+) T lymphocytes of African HIV-infected patients. M- and env-stimulated IFN-gamma-producing CD8(+) T cells were reduced in African participants and not increased by preincubation with alpha IL-10 monoclonal antibody. This is the first set of data that has reported immune activation in rural Africa by single-cell analysis of cytokine production. These results help in defining the immunologic background to be considered in the design of therapeutic and vaccine-based approaches to HIV infection in an African setting.

Black People↗

PubMed1991

A sensitive and efficient induction system for murine IgE. Single cell analysis at the clonal level.

A culture system is described which permits the analysis of IgE expression by single murine cells within clones of B cells. The system is based on the use of a CB5.1 stroma cell line as a feeder which optimally supports the IL-4-induced switch to IgE of LPS-stimulated B cells in culture. In this system 100 U/ml IL-4 induces the switch to IgE, in 3-5% of B cells and the switch frequency to IgG1 was as high as 2%. Five ng IgE or 12 ng IgG1 were produced per clone containing on average 13-15 PFC. The detection of single IgE secreting B cells was possible due to two newly developed, highly specific rat anti-mouse IgE antibodies used in a sandwich-ELISA. The frequency of IgE-secreting B cells was enhanced 2.5 times when the fibroblastoid CB5.1 cells rather than thymocytes were used as feeder cells. CB5.1 cells supported the differentiation of B cells to IgM-PFC almost as well as rat thymocytes (which have, to date, been used as the standard feeder layer) whereas the amount of secreted IgG1 was about 3 times lower than in thymocyte cultures. Optimal switching to IgE occurred at concentrations of IL-4 which were 10-fold lower than that required for IgG1 expression, a situation quite opposite to that observed in the rat thymocyte-supported culture system. In confirmation of established data the switch of B cells to IgE or IgG1 occurred randomly. The advantages of CB5.1 cells as feeder cells are (1) the use of a homogeneous and defined cell line, (2) their limited release of defined lymphokines (IL-6 and GM-CSF), and (3) the low degradation and consumption of cytokine factors. The combination of the CB5.1 cell line with a highly specific IgE ELISA assay made it possible to analyse the appearance of IgE producing cells within a developing B cell clone.

Animals↗