FACS - based isolation of human eosinophils allows purification of high quality RNA (original) (raw)
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Purification of functional eosinophils from human bone marrow
Journal of Immunological Methods, 2013
Eosinophils are granulocytic leukocytes that are best known for their involvement in host immune defense and pathologic states. More recently, they have also been shown to play a role in regulation of murine plasma cell homeostasis in the bone marrow, which prompted our investigation of human bone marrow eosinophils. However, effective methods to isolate eosinophils from human bone marrow thereby allowing comparisons with circulating eosinophils have not yet been described. Herein we describe the development of a novel, cost effective protocol for the purification of eosinophils from human bone marrow that allows us to obtain bone marrow eosinophils of near 100% purity after an 8-day culture system. Furthermore, we demonstrate that bone marrow eosinophils have characteristics similar to blood eosinophils, including the expression of IL-5Rα, the presence of eosinophil-specific granules, and similar activation kinetics upon phorbol myristate acetate and high-dose IL-5 stimulation. While migratory responses toward the chemokine CXCL12 differed between purified bone marrow and freshly isolated blood eosinophils, migratory responses were similar upon comparison of bone marrow eosinophils with blood eosinophils cultured ex vivo for 8 days prior to assay. Interestingly, a concurrent upregulation of CXCR4 expression was not observed in these cultured blood eosinophils. Taken together, we have overcome the existing challenges to the study of bone marrow eosinophils through our novel strategy for cell purification and have thus enabled future investigations of these cells and their role(s) in human health and disease.
The Journal of Immunology, 1999
Despite increasing interest, very little information exists regarding gene regulatory mechanisms employed by eosinophils. This largely stems from the difficulty in transfecting these primary cells. In this study, we demonstrate that peripheral blood eosinophils (PBEos) can be successfully transfected with both GM-CSF cDNA and mRNA and reporter constructs by particle-mediated gene transfer. The transfection efficiency was 1.2% based on green fluorescent protein-positive cells. Promoter studies revealed CMVdriven expression vectors were initially active but rapidly quenched, while viral long terminal repeats had greater activity, indicating that certain viral constructs may be relatively poor to direct the production of transgenic proteins in PBEos. Exogenous GM-CSF mRNA was readily delivered and detected by Northern blot, permitting determination of its t 1/2 in the absence of transcriptional poisons. These data show PBEos rapidly degraded GM-CSF mRNA with a t 1/2 of 8 min. Mutant GM-CSF mRNAs, lacking the AUUUA motifs, were more stable, but were still rapidly degraded, suggesting the existence of accessory, destabilizing elements. We were able to measure minute amounts of intracellular GM-CSF after the transfection of mutant GM-CSF mRNA, but extracellular cytokine was below the sensitivity of our ELISA. However, the presence of secreted GM-CSF was established by in vitro, survival bioassay. In conclusion, the existence of this new technology should allow detailed studies of eosinophil-specific transcriptional and posttranscriptional regulation.
Eosinophils from the blood of normal individuals were purified by centrifugation over discontinuous Percoll gradients. Eosinophil suspensions were obtained with a mean purity of 96% and a mean recovery of 64% (n = 19). When incubated with phorbol-myristate acetate, eosinophils consumed twice as much oxygen as did neutrophils from the same donors. With serum-treated zymosan, 70% and 100% of the maximal oxidative response (i.e. the response to phorbol-myristate acetate) was obtained with eosinophils and neutrophils, respectively. The calcium ionophore A23187 is a weak stimulus that triggered only 2-5% of the eosinophil and 10% of the neutrophil oxidative capacity. The response of both cell types to formyl-methionyl-leucyl-phenylalanine (fMLP) was rapid, with a maximum after 3 min. The magnitude of this eosinophil reaction was half that of neutrophils. Although the activities of the granule enzymes ,B-glucuronidase and arylsulphatase were 2-5 and 6 times higher in eosinophils than in neutrophils, respectively, the exocytosis of these enzymes in response to various stimuli was lower in eosinophils. The high yield of eosinophils from our separation method enabled us to prepare eosinoplasts by centrifugation of eosinophils over discontinuous Ficoll gradients that contained cytochalasin B. Eosinoplasts are plasma membrane vesicles derived from eosinophils, filled with cytoplasm but devoid of granules and nucleus. The eosinoplasts contained 30% of the cytoplasm and plasma membrane present in intact eosinophils. Eosinoplasts still possessed a functionally intact oxidase enzyme that could be stimulated with various stimuli. Therefore, eosinoplasts may provide a valuable tool to study separately the role of the oxidase products and that of the granule contents in eosinophil functions.
Identification of human eosinophils in whole blood by flow cytometry
Methods in molecular biology (Clifton, N.J.), 2014
Identifi cation of eosinophils in whole blood samples by fl ow cytometry is often problematic. There are usually only a low number and percentage of cells that may be detected, and it may be diffi cult to discriminate eosinophils from other granulocytes. Here, we propose a simple approach using the eosinophil's intrinsic autofl uorescence properties, combined with detection of CCR3 expression, to reliably identify eosinophils in a mixed leukocyte population.
BMC Research Notes, 2020
Objective Little has been reported regarding the reliability of methods for the purification of human blood eosinophils. We retrospectively reviewed our experience with 350 consecutive eosinophil isolations. Results Between January 2014 and December 2018, we conducted 350 eosinophil purifications from 83 donors. Absolute eosinophil count (AEC), calculated from hospital complete blood counts when available (n = 289), ranged from 32 to 1352 eosinophils/µL ($$\bar{x} \pm {\text{SD}}$$x¯±SD: 179 ± 136/µL). Eosinophil yields ranged from 0.4 to 24.4 million cells per 20 mL of blood drawn ($$\bar{x} \pm {\text{SD}}$$x¯±SD: 3.1 ± 1.9 million eosinophils) with > 98% purity. Comparing AEC to actual yield, recovery was 87% ± 29% ($$\bar{x} \pm {\text{SD}}$$x¯±SD) and AEC strongly correlated with yield. To explore the reproducibility of yield, a subsequent analysis was limited to those donors drawn ≥ 3 times (N = 35), and there was no difference in the average coefficient of variation for yi...
A simple method to obtain pure granule-rich eosinophil fragments (cytosomes) from normal human blood
Journal of Immunological Methods, 1985
This paper describes a simple, rapid and reproducible method to obtain pure granule-rich eosinophil fragments (cytosomes) with a high yield from normal human blood. The method is based on the treatment of whole blood with saponin and subsequent purification of the cytosomes on Percoll gradient. The enzymatic analysis of the cytosomes shows that the content of 3 granular enz~(mes is of the same order of magnitude already reported by others for intact eosinophils. This finding suggests that the cytosomes can be employed as starting material for studying the content of the granules or for the isolation of the granular components. The advantages offered by this method over those currently used to obtain eosinophil granules are discussed.
Eosinophils: multifaceted biological properties and roles in health and disease
Immunological Reviews, 2011
Summary: Eosinophils are leukocytes resident in mucosal tissues. During T‐helper 2 (Th2)‐type inflammation, eosinophils are recruited from bone marrow and blood to the sites of immune response. While eosinophils have been considered end‐stage cells involved in host protection against parasite infection and immunopathology in hypersensitivity disease, recent studies changed this perspective. Eosinophils are now considered multifunctional leukocytes involved in tissue homeostasis, modulation of adaptive immune responses, and innate immunity to certain microbes. Eosinophils are capable of producing immunoregulatory cytokines and are actively involved in regulation of Th2‐type immune responses. However, such new information does not preclude earlier observations showing that eosinophils, in particular human eosinophils, are also effector cells with proinflammatory and destructive capabilities. Eosinophils with activation phenotypes are observed in biological specimens from patients wit...
Direct flowcytometric analysis of eosinophils using a whole blood staining technique
Allergology International, 2001
Background: Flowcytometric analysis of purified eosinophils requires several hours to accomplish due mainly to purification of cells; moreover, it requires more than 20 mL blood and expensive reagents. The aim of the present study was to develop a method of direct flowcytometric analysis for eosinophils using whole blood. Methods: Peripheral blood obtained from five healthy individuals (mean age 42 years) and 10 patients with eosinophilia (mean age 40.3 years) were used for analysis. We stained antigens (CD9 or CD16) and fixed cells with parabenzoquinone (PBQ) or paraformaldehyde (PFA) after hemolyzation followed by treatment with N-octyl-β-glucopyranoside (OG). Results: On comparison of forward scatter with side scatter dot plots among samples treated with hemolyzation alone, PBQ fixation and PFA fixation, PBQ fixation showed the best results in discriminating eosinophils from other leukocyte fractions on the cytogram. Following fixation and permeabilization of cells, EG2, a secretory form of eosinophil cationic protein, was stained as an intracellular antigen. Flowcytometric analysis for EG2 showed a high positivity rate only in the eosinophil fraction. There were no differences in EG2 positivity or mean fluorescence intensity (MFI) between heparinized and EDTA-treated blood. Comparison of samples treated with OG at 6.0 and 7.4 mg/mL showed that the latter had a higher MFI for EG2 without significant change in the positivity rate. Conclusions: The findings show that intra-and extracellular properties of eosinophils can be analyzed with whole blood using PBQ fixation and OG treatment at a concentration of 7.4 mg/mL. Direct flowcytometric analysis of eosinophils saves time and requires only a small amount of blood, both of which are advantageous for patients and laboratory workers.