Cross-presentation of tumour antigens by human induced pluripotent stem cell-derived CD141(+)XCR1+ dendritic cells.

Monocyte-derived dendritic cells (moDC) have been widely used in cancer immunotherapy but show significant donor-to-donor variability and low capacity for the cross-presentation of tumour-associated antigens (TAA) to CD8(+) T cells, greatly limiting the success of this approach. Given recent developments in induced pluripotency and the relative ease with which induced pluripotent stem (iPS) cell lines may be generated from individuals, we have succeeded in differentiating dendritic cells (DC) from human leukocyte antigen (HLA)-A(*)0201(+) iPS cells (iPS cell-derived DC (ipDC)), using protocols compliant with their subsequent clinical application. Unlike moDC, a subset of ipDC was found to coexpress CD141 and XCR1 that have been shown previously to define the human equivalent of mouse CD8α(+) DC, in which the capacity for cross-presentation has been shown to reside. Accordingly, ipDC were able to cross-present the TAA, Melan A, to a CD8(+) T-cell clone and stimulate primary Melan A-specific responses among naïve T cells from an HLA-A(*)0201(+) donor. Given that CD141(+)XCR1(+) DC are present in peripheral blood in trace numbers that preclude their clinical application, the ability to generate a potentially unlimited source from iPS cells offers the possibility of harnessing their capacity for cross-priming of cytotoxic T lymphocytes for the induction of tumour-specific immune responses.

[A pocket pRb mutation induces the increase in its affinity to E2F4 coupled with activation of muscle differentiation].

Co-ordination of proliferation and differentiation in cells committed to muscle fate requires the interaction of the retinoblastoma gene product (pRb) with transcription factors of the E2F family. pRb has different affinities for distinct E2Fs, however, the mechanism involved in pRb-E2Fs interaction has not been completely investigated. We have found that pRb carrying a small deletion at the end of the T antigen binding region (deltaS/N), and unable to interact with large T antigen, could induce acute cell cycle block, stable prolongation of the cell cycle in G0/G1 and G2/M phases and suppression of the growth of tumor cells. The deltaS/N showed increased affinity for E2F4, bound hyperphosphorylated forms of E2F4 and induced its nuclear compartmentalization. The ability of deltaS/N to form complexes with E2F4 on DNA was associated with an increase in formation of "free" E2F4 and transsuppression of the specific reporter through preferential binding to E2F4 but not t o E2F1. Stable expression of deltaS/N in multi-potent fibroblasts promoted early muscle commitment. The results obtained suggest that a mutation in the T antigen binding region may increase in affinity of the pRb for E2F4 combined with activation of muscle differentiation.

Clinical stem cell therapies for severe autoimmune diseases.

Severe autoimmune diseases (ADs) are a major source of disability and reduced quality of life and may result in shortened life expectancy, particularly in treatment-resistant patients. For several decades, allogeneic and autologous haematopoietic stem cell transplantation (HSCT) has been the focus of scientific investigation as a potential means of delivering 'one-off' intensive treatment in severe ADs. Improvements in the clinical safety of HCST were followed by its increasing use in recent years as an experimental treatment for severe and resistant ADs. European and North American registries have accumulated between one and two thousand procedures. Retrospective analyses and prospective studies have demonstrated the feasibility, safety and initial efficacy data in various ADs. Profound cell biological changes induced by HSCT leading to stabilization or reversal of organ damage have been characterized. These have also shed light on basic disease mechanisms and support investigation of more specific cellular therapy in ADs. There is clear potential for harnessing a profound immunological effect through HSCT. However, there is a need for an ongoing balance against evolving non-transplant treatments for ADs. Ideally, these issues should be resolved in phase III studies, in which HSCT approaches are compared with the best comparator.

Stem cell-related therapies for vascular diseases.

The therapeutic potential of 'adult' or at least non-embryonic stem cells and their progeny has developed gradually over the past half century as a consequence of the wealth of knowledge derived from stem cell research. Translational research coupled with clinical trials and derived from basic research has led the way to the clinic. This commenced with the use of haematopoietic stem cell transplantation (HSCT), to treat haematological malignancies, to be followed by the most recent clinical trials to treat a variety of coronary and peripheral artery diseases. Stem cells and their progeny isolated from bone marrow or blood appear to exert an ameliorating effect in certain vascular disorders. Although promising, some of these treatments remain controversial and further research and, where indicated, appropriately powered trials are required to confirm the safety and determine the efficacy of these novel therapies.

Mobilization of endothelial progenitor cells into the circulation in burned patients.

BACKGROUND: Bone marrow-derived endothelial progenitor cells (EPCs) have been detected in the peripheral blood of patients following thermal injury. EPCs migrate to sites of active neovascularization in response to mediators released after trauma, contributing to wound healing. The aim was to characterize levels and kinetics of EPCs in burned patients, then relate these to key mobilizing factors, vascular endothelial growth factor (VEGF) and the chemokine (C-X-C motif) ligand 12 (CXCL 12), and compare them with those in healthy subjects. METHODS: The study included 19 adult patients with superficial or full-thickness burns and 50 blood donor volunteer controls. EPCs, identified by cell surface markers CD45(dim/-), CD133+, CD144+ and VEGF receptor 2, were quantified by four-colour flow cytometry. Plasma VEGF and CXCL12 were measured using enzyme-linked immunosorbent assay. RESULTS: Burned patients showed a rapid rise in EPC levels within 24 h, a ninefold increase compared with controls, returning to basal levels by 72 h. Body surface area burned correlated strongly with the degree of mobilization. EPC levels correlated significantly with rises in plasma VEGF and CXCL12. CONCLUSION: Thermal injury induced a rapid rise in EPCs that was proportional to the extent of the burn and significantly correlated with levels of angiogenic cytokines. Such cytokines may be used to stimulate EPCs as a future therapeutic target in burned patients.

5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies.

BACKGROUND AND OBJECTIVES: Mesenchymal stem/progenitor cells (MSCs) are multipotent progenitors that differentiate into such lineages as bone, fat, cartilage and stromal cells that support haemopoiesis. Bone marrow MSCs can also contribute to cardiac repair, although the mechanism for this is unclear. Here, we examine the potential of MSCs from different sources to generate cardiomyocytes in vitro, as a means for predicting their therapeutic potential after myocardial infarction. MATERIALS AND METHODS: Mesenchymal stem/progenitor cells were isolated from the perivascular tissue and Wharton's jelly of the umbilical cord and from cord blood. Their immunophenotype and differentiation potential to generate osteoblasts, chondrocytes, adipocytes and cardiomyoxcytes in vitro was compared with those of bone marrow MSCs. RESULTS: Mesenchymal stem/progenitor cells isolated from umbilical cord and cord blood were phenotypically similar to bone marrow MSCs, the exception being in the expression of CD106, which was absent on umbilical cord MSCs, and CD146 that was highly expressed in cord blood MSCs. They have variable abilities to give rise to osteoblasts, chondrocytes and adipocytes, with bone marrow MSCs being the most robust. While a small proportion (approximately 0.07%) of bone marrow MSCs could generate cardiomyocyte-like cells in vitro, those from umbilical cord and cord blood did not express cardiac markers either spontaneously or after treatment with 5-azacytidine. CONCLUSION: Although MSCs may be useful for such clinical applications as bone or cartilage repair, the results presented here indicate that such cells do not generate cardiomyocytes frequently enough for cardiac repair. Their efficacy in heart repair is likely to be due to paracrine mechanisms.

Quantification of circulating cell-free plasma DNA and endothelial gene RNA in patients with burns and relation to acute thermal injury.

BACKGROUND: Major burn represents a multi-system insult to the human body. Despite improvements in mortality and morbidity, reliable predictors of outcome are lacking. Raised levels of cell-free nucleic acids have been detected in various pathological processes including burns. We quantified circulating nucleic acids as potential objective measures of burn severity with predictive and prognostic value. METHODS: Expression of endothelial specific cell-free mRNA and cell-free DNA were measured in plasma of 19 burn patients at days 1-3 and week 10 following acute thermal injury and in 19 healthy controls by real-time quantitative PCR. RESULTS: Expression of endothelial specific mRNA was higher in burn patients compared to controls (p<0.001). DNA levels were significantly higher in the burn population in the first 48 h following injury. Plasma RNA and DNA levels related to %TBSA burn in the first 24h and to the levels of circulating endothelial progenitor cells. CONCLUSIONS: We show that plasma levels of endothelial specific mRNA and DNA are elevated acutely following burns, and relate to severity in terms of %TBSA burnt.

Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation.

The COBE Spectra is used to volume/red blood cell (RBC) deplete BM before transplantation or cryopreservation. We have audited our results to identify the effect of transit time, refrigerated storage, age and cellular composition on mononuclear cells (MNC) and CD34+ cell recoveries, volume/RBC depletion and neutrophil engraftment. In total, 88 consecutive collections from autologous (n = 25) and allogeneic (n = 63) donors were included. The mean collection volume was 1250 +/- 398 ml with RBC content of 341 +/- 113 ml. The MNC and CD34+ cell recoveries were 83.3 +/- 18.5 and 88.1 +/- 18.9%, respectively, volume depletion was 88.2+/-4.4% and RBC depletion 98.3 +/- 1.8%. Neutrophil engraftment was achieved in 20.1 +/- 6.4 days. Factors affecting MNC and CD34+ cell recoveries were transit time (P = 0.0060), overall age (P < 0.0210) and MNC/CD34+ cell concentrations (P < 0.0313). The presence of crenated RBC also reduced CD34+ cell recovery (P = 0.0028). Refrigerated storage did not adversely affect cell recovery (P > 0.8161) or neutrophil engraftment (P = 0.8959). This study demonstrates that time in transit, overall age, MNC and CD34+ cell concentrations and RBC condition were important factors affecting processing. RBCs show artefacts soon after collection at ambient temperatures and these may interfere with the separation and collection of MNC/CD34+ cells. Refrigeration at 4-6 degrees C during transit and storage may reduce formation of RBC artefacts and maximize MNC and CD34+ cell recoveries.

High-resolution two-dimensional electrophoretic analysis of acidic, basic and surface proteins of mouse neutrophils.

The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 20 proteins detected) and with fluorography or autoradiography after [35S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of 125I associated with actin) yielded more than 20 major 125I-labelled proteins on high-resolution electrophoretic maps. The major 125I-labeled protein (Mr approximately 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [35S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.

Gp130-signaling synergizes with FL and TPO for the long-term expansion of cord blood progenitors.

We investigated the effect of a new fusion protein of IL-6 and the soluble IL-6R, H-IL-6, on the long-term ex vivo expansion of hematopoietic progenitors derived from AC133+cord blood cells. H-IL-6, which acts on both IL-6Ralpha-positive and IL-6Ralpha-negative cells, effectively synergized with FL and TPO with or without SCF for the propagation of primitive progenitors. However, IL-6 showed a greater synergistic effect with FL and TPO than H-IL-6 for long-term progenitor propagation. During the first 6 weeks of culture under stroma-free serum-containing conditions, IL-6 induced a 1.96 +/- 0.64-fold higher expansion of nucleated cells, a 2.28 +/- 0.33-fold higher expansion of CD34+ cells and a 2.74 +/- 0. 28-fold higher expansion of CD34+ AC133+ cells than H-IL-6 in combination with FL and TPO. The propagation of week 6 CAFC was up to four-fold higher in the presence of IL-6 than with H-IL-6. While the expansion of CD34+ and CD34+ AC133+ cells dropped after 5-7 weeks in the stroma-free cultures with FL, TPO and H-IL-6, a sustained expansion for 12 weeks was obtained in the presence of FL, TPO and IL-6. Stroma-contact greatly enhanced the progenitor expansion induced by FL and TPO or FL, TPO and H-IL-6 although the highest proliferation was again obtained in the presence of IL-6. In contrast, the presence of SCF resulted in increased differentiation. Since the majority of primitive progenitors are proposed to be IL-6Ralpha-negative, the results suggest that the synergistic effect of IL-6 is mediated by accessory cells, which have been more effectively stimulated by IL-6 than by the fusion peptide, H-IL-6, in this culture system.

Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34.

Monoclonal antibodies of the CD34 class all recognize a monomeric cell surface antigen of approximately Mr 110,000 which is selectively expressed on human hemopoietic progenitor cells. This structure can be readily surface-labeled with [125I]actoperoxidase and by periodate-[3H]borohydride, but it labels only weakly with [35S]methionine, [35Sl]cysteine, 3H-amino acids, or 3H-mannose, even after prolonged labeling periods. However, the antigen is more efficiently labeled by [3H]glucosamine. Lectin binding studies, sensitivity to certain glycosidases, and gel filtration analysis of glycans released by alkaline hydrolysis indicate that this glycoprotein contains several complex-type N-linked glycans as well as several highly sialylated O-linked glycans. Western blotting experiments show that various CD34 antibodies fail to efficiently detect desialylated and/or de-N-glycosylated forms of the antigen. Experiments involving the use of tunicamycin, together with metabolic labeling studies, strongly suggest that this structure "turns over" very slowly in vivo. The CD34 antigen is not detectably labeled by 32P-phosphate in vivo, nor are immune complexes containing it associated with phosphokinase activity in vitro. Sequential immunoprecipitation and Western blotting studies indicate that this antigen is not a member of the leukosialin/sialophorin family despite the fact that these molecules share several structural similarities. Partial amino acid analysis of highly purified CD34 antigen revealed no significant sequence similarity with any previously described structures.

Detection of additional JH/BCL2 translocations in follicular lymphoma.

In vitro enzymatic amplification and direct sequencing were used to detect and characterize t(14;18) recombination junctions in peripheral blood and bone marrow mononuclear cell preparations from patients with follicular lymphoma in remission. Samples from 24/44 patients were found to be positive for translocations involving the major breakpoint region of the BCL2 gene. In samples from seven patients two distinct t(14;18) translocations were shown to be present simultaneously; in one case the second translocation involved the minor cluster region of the BCL2 gene. Biopsy tissue obtained earlier in the course of the disease was available from five of these patients and was shown to contain one of the translocations in each case, but both translocations in only one. Further remission blood and bone marrow samples from the group were also examined. This led to the detection of both translocations in separate samples obtained at different times in a total of four out of the seven cases. In two of the remaining three patients the second translocation could not be amplified from further samples, but in both cases the search led to the identification of a third translocation, again only detectable in a single sample. These findings demonstrate that JH/BCL2 translocations can occur more than once during the course of follicular lymphoma. They suggest that biclonal follicular lymphoma may be more common than has previously been recognized but also raise the possibility that the translocation arises sporadically in the normal lymphoid cells of this group of patients.

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells.

Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.

An improved negative immunomagnetic selection strategy for the purification of primitive hemopoietic cells from normal bone marrow.

An improved negative immunomagnetic selection strategy has been devised for the enrichment of primitive hemopoietic cells using the high proliferative potential colony-forming cell (HPP-CFC) assay as an index of stem cell purification. Immunomagnetic selection was carried out using goat anti-rat conjugated M-450 Dynabeads and a cocktail of rat monoclonal antibodies directed against lineage antigens expressed on B-lymphocytes (B220), neutrophils and activated macrophages (7/4), differentiating erythroid cells (YW 25.12.7), and T-lymphocyte subsets (Lyt-2 and L3T4). This negative selection strategy results in the highly reproducible enrichment of HPP-CFC with negligible loss of HPP-CFC at the immunomagnetic selection step. A 30-fold enrichment of HPP-CFC stimulated by interleukin 3 (IL-3) plus colony-stimulating factor (CSF-1), or interleukin 1 alpha (IL-1 alpha) plus IL-3 plus CSF-1, is obtained with simultaneous resolution of HPP-CFC from progenitor cells of low proliferative potential responsive to CSF-1 alone (LPP-CFC). Flow cytometric analysis of these lineage-negative cells reveals that they almost exclusively exhibit the light-scattering characteristics of blast cells and the morphology of a candidate hemopoietic stem cell. Positive fluorescence-activated cell sorting of immunomagnetically pre-enriched normal bone marrow cells using wheat germ agglutinin yields cell preparations with a cloning efficiency of up to 45% and a HPP-CFC content of 20%.

Adhesion receptors are differentially expressed on developing thymocytes and epithelium in human thymus.

The thymic microenvironment consists of a network of interrelated cells of epithelial, fibroblastic, endothelial, and hemopoietic origin. Within this environment, the development of specific T-lymphocyte subpopulations partially depends on the selective interaction of T-cell precursors with such cells. Human thymic epithelial cell strains, generated with a defective retroviral vector containing simian virus 40 (SV40) large T antigen and the neomycin resistance gene or by transfection with an SV40 plasmid defective in the origin of replication, provide useful tools for understanding the mechanisms contributing to the control of T-cell maturation. Because interepithelial, epithelial-macrophage, and lymphocyte-epithelial cell interactions are important for thymocyte differentiation, the distribution of integrin and nonintegrin adhesion receptors on these cells and on developing thymocytes in vivo and in vitro has been examined in detail. Our results indicate that the transformed human thymic epithelial cell strains express the common very late antigen (VLA)-beta 1 receptor and unique alpha chains VLA-2, VLA-3, and VLA-6. The cells are also positive for LFA-3 and ICAM-1 and weakly express beta 3, beta 4, and VNR alpha. They do not express the Leu-cellular adhesion molecules (CAM). This phenotypic profile on cultured thymic epithelium generally corresponds to the distribution of integrin and other receptor molecules on thymic epithelial cells in tissue sections. The majority of thymocytes also express the integrin VLA-beta 1 and -beta 2 chains as well as VLA-4, VLA-6, and LFA-1 alpha(L). Three-color flow cytometric analyses show differential levels of expression of these adhesion receptors on human thymocyte subsets. Taken together with the immunohistochemical localization of extracellular matrix molecules, these studies suggest that both the distribution of receptor-ligand pairs and the level of expression of adhesion molecules may influence T-cell development within the thymus.

Preparation and surface labeling of murine eosinophils.

Eosinophilic polymorphonuclear leukocytes were isolated from the peritoneal cavity of BALB/c mice infected with the parasite Mesocestoides corti. Approximately 4 X 10(7) eosinophils (purity, 50%) could be harvested from each mouse. A high yield and purity of eosinophils was obtained from the peritoneal cells of infected male BALB/c mice using density centrifugation on a gradient of slightly hypotonic colloidal silica sol (Percoll). After initial irradiation of the mice to lower the lymphocyte contamination, subsequent density gradient (and where necessary sedimentation velocity) centrifugation yielded 10(8) eosinophils (purity > 95%) from six to eight mice. It was also possible to isolate small numbers of eosinophils (2 X 10(4) cells/minute, purity > 99%) without irradiating the mice. This could be achieved by separating the density gradient purified peritoneal cells by light-scatter on a Becton-Dickinson cell sorter (FACS II). Analysis of proteins extracted from eosinophils using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a group of high molecular weight proteins (bwtween 250K and 160K) which were not as distinctive in the neutrophil profile. Surface labeling was performed, before the cell separation by using 125I and 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. Only five 125I-labeled proteins were detected initially (all with apparent molecular weights > 50,000). No 125I appeared to be associated with actin under the conditions used for surface labeling. Four of the eosinophil surface labeled proteins corresponded to surface labeled proteins on neutrophils, but the major surface component of the eosinophils (MW 79,000) appeared to be smaller than the major neutrophil protein (MW 90,000).

An improved panning technique for the selection of CD34+ human bone marrow hematopoietic cells with high recovery of early progenitors.

The human hematopoietic pluripotent repopulating "stem cell" is thought to be present within a minor subpopulation of CD34+ cells. This has not been definitively shown, although the more primitive CD34+ cell subset contains precursors for all lymphoid and nonlymphoid cell lineages. When purifying CD34+ cells, it is important to recover these early progenitors, which are more strongly immunoadsorbent to the separation devices. Using a commercialized panning system (AIS CELLector flasks), we observed that a high degree of purity requires a thorough washing procedure so that cells not binding or weakly binding to CD34 antibodies are removed. High recoveries can be obtained if the adherent cells are then efficiently detached by a 2-hour incubation in culture medium without added cytokines. In this way, we can routinely obtain 93.5 +/- 3.4% purity of CD34+ cells with a 74% yield of the multipotent colony-forming units (CFU-GEMM). Complete recovery of the putative "stem cell," or at least the early progenitor cell compartment (CD34+ CD38low/- CD34+ Thy-1+ cells), is also obtained. More than 30% of these cells can generate day-14 colonies in vitro. Comparable results were obtained when the separation was scaled up for clinical application using appropriate large-scale devices. The various incubation times of the procedure can be easily adjusted to the work schedule. This renders the procedure easy to handle, efficient, safe, and, because the cells can be observed under light microscopy, easy to control.

The characterisation of monoclonal antibodies against haemopoietic cells: comparison of an immunoperoxidase method with fluorescence activated cell sorting.

Nucleated cells from normal human peripheral blood and bone marrow have been analysed with 2 rat monoclonal antibodies of known specificity, one (YAML 555.6.6) directed against the P28,33 complex (anti-HLA-DR), and the other (YAML 501.4.4) directed against leucocyte common antigen (anti-LCA). The patterns of reactivity with an indirect immunoperoxidase method on fixed smeared cells were in close agreement with those obtained with a fluorescence activated cell sorter. A further new monoclonal antibody of unknown antigen specificity (YAML 537.2) reacts with an intracellular antigen present in neutrophils and their precursors from the promyelocyte stage onwards, megakaryocytes, and a proportion of monocytes, but not with eosinophils, nucleated red cells or lymphocytes. This reactivity could not be demonstrated using the fluorescence activated cell sorter. The immunoperoxidase method allows the identification of individual positive and negative cells and therefore provides a method of identifying minor reactive and non-reactive cell populations in a heterogeneous cell sample such as normal bone marrow. Cytoplasmic binding sites can be differentiated from membrane binding.

Isolation of mouse bone marrow neutrophils by light scatter and autofluorescence.

Murine bone marrow and blood cells have been analyzed and fractionated using an automated FACS II cell sorter. Using visible light scattered in the direction of (0 degrees) and perpendicular to (90 degrees) the laser beam it was possible to enrich for neutrophils (84%), immature myeloid cells (47%), and monocytes (78%). The enrichment for neutrophils was improved to 92% by using the light scattered by ultraviolet laser light (ca.360 nm). The autofluorescence at these wavelengths proved useful for obtaining further enrichment (to 97%). Indeed, three parameter sorting with 0 degrees and 90 degrees light scatter as well as autofluorescence also allowed the separation of lymphocytes (95%) and immature myeloid cells (89%). The same procedures could be applied for the isolation of neutrophils from mouse peripheral blood.