Kinetically Limited Differential Centrifugation as an Inexpensive and Readily Available Alternative to Centrifugal Elutriation

Tan, Jinwang; Lee, Byung-Doo; Polo‐Parada, Luis; Sengupta, Shramik

doi:10.2144/0000113853

Cited by 11 publications

(8 citation statements)

References 24 publications

(17 reference statements)

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“…To isolate leukocytes, blood was layered onto Histopaque-1083 (Sigma) for density gradient centrifugation. Leukocytes were collected between the plasma layer and the pellet containing red blood cells (RBCs) and extracellular bacteria ( 34 ). Isolated leukocytes were then washed well and resuspended in Fcγ block for flow cytometric analysis or lysed in sterile water to quantify cell-associated CFU.…”

Section: Methodsmentioning

confidence: 99%

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection

et al. 2015

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The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.

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Section: Methodsmentioning

confidence: 99%

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection

et al. 2015

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“…Separation of the bacterial fraction from the platelet samples was performed using the density difference between platelets (1.05−1.07) and bacteria (>1.10). 43 This separation of bacterial fraction from platelets was performed by adding Histopaque-1083 and using centrifugation, as shown in Figure S4. The quantitative analysis of the Gram-negative bacteria in the platelet samples was performed via the reaction of the separated bacterial fraction with anti-OMP antibodies through flow cytometry.…”

Section: Resultsmentioning

confidence: 99%

“…The isolated anti-OMP antibodies against Gram-negative bacteria and anti-LTA antibodies against Gram-positive bacteria were employed for the detection of bacterial contamination in platelets. Separation of the bacterial fraction from the platelet samples was performed using the density difference between platelets (1.05–1.07) and bacteria (>1.10) . This separation of bacterial fraction from platelets was performed by adding Histopaque-1083 and using centrifugation, as shown in Figure S4.…”

Section: Resultsmentioning

confidence: 99%

Rapid Analysis of Bacterial Contamination in Platelets without Pre-Enrichment Using Pig Serum-Derived Antibodies

Bong

Park

Sung

et al. 2021

ACS Appl. Bio Mater.

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As the shelf life of platelets collected from donated blood is very short, approximately 5 days, the determination of bacterial contamination in platelets has become necessary. In this study, rapid analysis of Gram-positive and Gram-negative bacterial contamination in platelet samples was presented without pre-enrichment using pig serum-derived antibodies against the outer membrane proteins (OMP) of Gram-negative bacteria and antibodies against lipoteichoic acid (LTA) on the surface of Gram-positive bacteria. The anti-OMP antibodies against Gram-negative bacteria were isolated using sequential incubation with (1) the modified Gram-negative bacteria ClearColi, which lacks lipopolysaccharide (LPS) on the outer membrane, and (2) the Gram-positive bacteriaBacillus subtilis to filter away nonspecifically bound proteins from ClearColi. The anti-lipoteichoic acid (LTA) antibodies against Gram-positive bacteria were isolated using sequential incubation with (1) the Gram-positive bacteriaB. subtilis and (2) the Gram-negative bacteria Escherichia coli BL21 to filter away nonspecifically bound proteins fromB. subtilis. The feasibility of using the antibodies isolated from pig serum against Gram-negative and Gram-positive bacteria was demonstrated using flow cytometry. Finally, detection of the contamination of platelets with Gram-negative and Gram-positive bacteria using the impedance immunosensor based on these isolated antibodies was successfully demonstrated.

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“…The bacteria in the collected blood were extracted by differential centrifugation according to the method described by Tan [ 30 ] with modifications. Briefly, three blood samples were centrifuged first at 200 × g for 10 min at 4℃; then, at 1,000 × g for 1 min and 1,500 × g for 30 sec to remove blood cells.…”

Section: Methodsmentioning

confidence: 99%

A two-component signal transduction system contributes to the virulence ofRiemerella anatipestifer

2018

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Similar to other studies of bacterial pathogens, current studies of the pathogenesis of Riemerella anatipestifer (RA) are focused mainly on in vitro culture conditions. To elucidate further the pathogenesis of RA in vivo, bacterial RNA was extracted from overnight tryptic soy broth cultures (in vitro) and from the blood of infected ducks (in vivo) for comparative RNA sequencing analysis. In total, 682 upregulated genes were identified in vivo. Among the upregulated genes, a signal transduction response regulator (ArsR) and a signal transduction histidine kinase (SthK) were predicted to be located on the same operon. A mutant was constructed by deletion of both of these genes. Duck infection tests showed that genes ArsR and SthK were related to the virulence of the pathogen in vivo. Differentially expressed genes identified by comparison of in vitro and in vivo conditions provided an insight into the physiological process of RA infection and provided an opportunity to identify additional virulence factors.

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Kinetically Limited Differential Centrifugation as an Inexpensive and Readily Available Alternative to Centrifugal Elutriation

Cited by 11 publications

References 24 publications

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection

Rapid Analysis of Bacterial Contamination in Platelets without Pre-Enrichment Using Pig Serum-Derived Antibodies

A two-component signal transduction system contributes to the virulence ofRiemerella anatipestifer

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