Separation methods for isolation of human polymorphonuclear leukocytes affect their motile activity

Sroka, Jolanta; Kordecka, Anna; Włosiak, Przemysław; Madeja, Zbigniew; Korohoda, W

doi:10.1016/j.ejcb.2009.05.005

Cited by 17 publications

(14 citation statements)

References 38 publications

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“…Whole-blood was chosen as a previous report showed that neutrophil isolation methods might affect cell motility (Sroka et al, 2009). In short, 600 μl of PBS with 1% human serum and recombinant HMGB1 or IL8 (R&D Systems, Minneapolis, MN; both supplied in preparations with endotoxin levels <1 EU/μg) was added to the basolateral side of a 3 μm pore-size polycarbonate transwell membrane (Coring incorporated, Corning, NY).…”

Section: Methodsmentioning

confidence: 99%

HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

Johansson

Snäll

Sendi

et al. 2014

Front. Cell. Infect. Microbiol.

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Extracellular High Mobility Group Box 1 (HMGB1) has been associated with acute and chronic inflammatory conditions. However, little is known about HMGB1 in necrotizing bacterial infections. We hypothesized that the local HMGB1 response is excessive in severe soft tissue infections (STIs), which are characterized by necrosis and hyperinflammation. To explore this, tissue biopsies were collected from patients with varying severity of Streptococcus pyogenes skin and STIs, including erysipelas, cellulitis, and necrotizing fasciitis. Tissue sections were immunostained for HMGB1, S. pyogenes, and inflammatory cell infiltrates and results quantified by acquired computerized image analysis (ACIA). HMGB1 expression increased in parallel to disease severity and was significantly higher in necrotizing fasciitis than in erysipelas (p = 0.0023). Confocal microscopy of sections co-stained for HMGB1 and cell markers revealed both extracellular and cytoplasmic HMGB1, the latter of which was found predominantly in macrophages. To further verify macrophages as main source of activation triggered HMGB1 release, human macrophages were infected with clinical S. pyogenes isolates. The results demonstrated infection triggered release of HMGB1. Dual staining's visualized HMGB1 in areas close to, but not overlapping, with neutrophils, indicating a potential chemotactic role. In vitro transmigration experiments showed a chemotactic effect of HMGB1 on neutrophils. The data furthermore provided in vivo support that HGMB1 may form immunostimulatory complexes with IL-1β. Taken together, the findings provide the first in vivo evidence that HMGB1 is abundant at the local site of severe bacterial STIs and its levels correlated to severity of infections; hence, indicating its potential value as a biomarker for tissue pathology.

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Section: Methodsmentioning

confidence: 99%

HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

Johansson

Snäll

Sendi

et al. 2014

Front. Cell. Infect. Microbiol.

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“…In addition, there is no consensus on the best technique for isolating granulocytes to achieve high purity. The methods applied for granulocyte isolation are diverse [29][30][31][33][34][35]53,[67][68][69][70], and their efficiency has been reported in different ways, such as cellular viability or cell isolation yield, cell functionality, number of target cells and contaminants after isolation, which makes it difficult to evaluate the methods for clinical application.…”

Section: Discussionmentioning

confidence: 99%

“…Granulocytes can be isolated from whole blood by different methods. The most commonly used method uses gradients of Percoll [29][30][31][32][33][34]. Isolation purity, viability and yield are important factors for evaluating each method and ensuring cellular integrity [29,[34][35][36].The noninvasive evaluation of acute inflammatory processes makes it possible to analyze molecular and cellular processes in vivo, usually based on targeting specialized cells such as macrophages, lymphocytes or granulocytes to the desired target [37].…”

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confidence: 99%

Methods of Granulocyte Isolation from Human Blood and Labeling with Multimodal Superparamagnetic Iron Oxide Nanoparticles

et al. 2020

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This in vitro study aimed to find the best method of granulocyte isolation for subsequent labeling with multimodal nanoparticles (magnetic and fluorescent properties) to enable detection by optical and magnetic resonance imaging (MRI) techniques. The granulocytes were obtained from venous blood samples from 12 healthy volunteers. To achieve high purity and yield, four different methods of granulocyte isolation were evaluated. The isolated granulocytes were labeled with multimodal superparamagnetic iron oxide nanoparticles (M-SPIONs) coated with dextran, and the iron load was evaluated qualitatively and quantitatively by MRI, near-infrared fluorescence (NIRF) and inductively coupled plasma mass spectrometry (ICP-MS). The best method of granulocyte isolation was Percoll with Ficoll, which showed 95.92% purity and 94% viability. After labeling with M-SPIONs, the granulocytes showed 98.0% purity with a yield of 3.5 × 10 6 cells/mL and more than 98.6% viability. The iron-loading value in the labeled granulocytes, as obtained by MRI, was 6.40 ± 0.18 pg/cell. Similar values were found with the ICP-MS and NIRF imaging techniques. Therefore, our study shows that it is possible to isolate granulocytes with high purity and yield and labeling with M-SPIONs provides a high internalized iron load and low toxicity to cells. Therefore, these M-SPION-labeled granulocytes could be a promising candidate for future use in inflammation/infection detection by optical and MRI techniques.Hard-to-reach or occult inflammation and infection processes are clinically challenging. Their clinical diagnoses involve biochemical and radiological examinations, but these methods can produce false negatives [11][12][13][14][15]. To determine the localization, extent and severity of inflammation, imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) can be performed but have limitations in the early stages of the disease, as they are dependent on morphological changes [16][17][18]. Accurate and early diagnosis of inflammation and infection helps reduce mortality and morbidity and increase drug treatment success [19][20][21].Proinflammatory effects help leukocytes migrate from the blood vessel to the site of injury, due to increased vascular permeability through stimulation by cytokines and interleukins. There is an initial accumulation of granulocytes, mainly neutrophils, in the inflamed area and later of lymphocytes and macrophages [22][23][24]. Granulocytes are the most abundant subset of leukocytes [25] and are produced in the bone marrow, originating from myeloid precursor cells and undergoing a maturing process before they are able to perform their functions, mainly phagocytosis [25,26]. They differ from other cells in that they have granules with proteolytic enzymes that fight microorganisms or other inflammatory agents [25,27]. Granulocytes are considered frontline cells in inflammation and infection processes [28]. In many cases, to detect inflammation/infection, it is necessary to label isolated granulocyte...

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“…They were composed from 70 sequential centroid position and processed by the program Mathematica. The scatter correlation diagrams of the trajectories, total displacement lengths and the Random walk analysis were prepared as described by Gail (Sroka, Kordecka, Włosiak, Madeja, & Korohoda, 2009). In addition, the total lengths of cell trajectory and displacement, percentage of migrating cells, as well as the movement and coefficient of movement efficiency were computed.…”

Section: Time-lapse Imaging Of D Discoideum Cellular Movementmentioning

confidence: 99%

Two‐photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment

Loukanov

2019

Microscopy Res & Technique

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In this study, a two‐photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil‐living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two‐photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.

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Separation methods for isolation of human polymorphonuclear leukocytes affect their motile activity

Cited by 17 publications

References 38 publications

HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

HMGB1 in severe soft tissue infections caused by Streptococcus pyogenes

Methods of Granulocyte Isolation from Human Blood and Labeling with Multimodal Superparamagnetic Iron Oxide Nanoparticles

Two‐photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment

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