scholarly journals Aptamer Cocktail to Detect Multiple Species of Mycoplasma in Cell Culture

2020 ◽  
Vol 21 (11) ◽  
pp. 3784 ◽  
Author(s):  
Quanyuan Wan ◽  
Xiaohui Liu ◽  
Zihua Zeng ◽  
Zhenghu Chen ◽  
Yanting Liu ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Cultured Cells ◽  
Detection System ◽  
Mycoplasma Contamination ◽  
Detection Model ◽  
Mycoplasma Detection ◽  
Infected Cells ◽  
Microplate Reader ◽  
Multiple Species

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

Endothelial cell cystine uptake and glutathione increase with N,N-bis(2-chloroethyl)-N-nitrosourea exposure

1992 ◽  
Vol 262 (3) ◽  
pp. L301-L304 ◽  
Author(s):  
S. M. Deneke ◽  
R. A. Lawrence ◽  
S. G. Jenkinson
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Culture Medium ◽  
Cultured Cells ◽  
Potent Inhibitor ◽  
Cell Types ◽  
Cell Culture Medium ◽  
Intracellular Accumulation ◽  
Pulmonary Endothelial Cells

Glutathione (gamma-glutamylcysteinylglycine, GSH) is an important cellular antioxidant. In typical cultured cell preparations GSH synthesis is limited by the availability of intracellular cysteine. Because extracellular cystine is the chief source of intracellular cysteine in cultured cells, increasing cystine transport can result in increased intracellular GSH. Depletion of GSH or exposure to oxidants has been shown to stimulate cystine transport in bovine pulmonary endothelial cells and other cell types. BCNU [N,N-bis(2-chloroethyl)-N-nitrosourea] is a potent inhibitor of glutathione reductase (GSSG-Red). We examined the effects of BCNU on cystine uptake by bovine pulmonary artery endothelial cells (BPAEC). We hypothesized that blocking GSSG-Red could result in increased cellular uptake of cystine to replenish decreases in GSH caused by oxidation. Levels of BCNU between 0.005 and 0.05 mM added to the cell culture medium inhibited GSSG-Red at 2, 4, and 24 h after addition. BCNU treatment resulted in concentration-dependent increases in both cystine uptake and GSH levels after 24 h of exposure. The increases in uptake were specific for cystine and glutamate and were sodium independent, suggesting induction of a xc(-)-like transport system. No intracellular accumulation of GSSG was measured nor was any significant depletion of GSH noted at any time of BCNU exposure.

scholarly journals HEPES inhibits the conversion of prion protein in cell culture

2011 ◽  
Vol 92 (5) ◽  
pp. 1244-1250 ◽  
Author(s):  
Karine Delmouly ◽  
Maxime Belondrade ◽  
Danielle Casanova ◽  
Ollivier Milhavet ◽  
Sylvain Lehmann
Keyword(s):  
Cell Culture ◽  
Prion Protein ◽  
Cell Biology ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Live Cells ◽  
Cell Culture Medium ◽  
Dependent Inhibition ◽  
Infected Cells ◽  
Significant Concentration

HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrPSc). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.

scholarly journals Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Cord C. Uphoff ◽  
Sabine-A. Denkmann ◽  
Hans G. Drexler
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Culture Medium ◽  
Detection Method ◽  
Mycoplasma Species ◽  
Mycoplasma Contamination ◽  
Cure Rate ◽  
Mycoplasma Detection ◽  
Additional Cell ◽  
Limited Period

A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

scholarly journals Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells

Metabolites ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 293 ◽  
Author(s):  
Veronika Kulikova ◽  
Konstantin Shabalin ◽  
Kirill Nerinovski ◽  
Alexander Yakimov ◽  
Maria Svetlova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Nicotinic Acid ◽  
Culture Medium ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Hek293 Cells ◽  
Vitamin B3 ◽  
Serum Free ◽  
Nicotinamide Riboside ◽  
Nicotinamide Mononucleotide

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

Presence of Trypanosoma cruzi antigen on the surface of both infected and uninfected cells in tissue culture

Parasitology ◽  
1980 ◽  
Vol 80 (1) ◽  
pp. 147-152 ◽  
Author(s):  
I. A. Abrahamsohn ◽  
J. K. Kloetzel
Keyword(s):  
Cell Culture ◽  
Tissue Culture ◽  
Trypanosoma Cruzi ◽  
Culture Medium ◽  
Culture Supernatant ◽  
Uninfected Cell ◽  
Cell Culture Supernatant ◽  
Supernatant Fluid ◽  
Cell Monolayers ◽  
Infected Cells

SummaryLLC-MK2 cell monolayers infected with Trypanosoma cruzi were shown by immunofluorescence to present parasite antigens on the surface of both parasitized and non-parasitized cells after completion of the first intracellular cycle and rupture of infected cells. The cell-culture supernatant fluid at this stage, as well as the supernatant fluid of parasites left overnight in culture medium were concentrated and contained antigen capable of binding to uninfected cell monolayers. The origin of this antigen, as well as its eventual role in the pathogenesis of Chagas' disease, are discussed.

The Use of Artemia Salina in Toxicity Testing

1992 ◽  
Vol 20 (2) ◽  
pp. 297-301 ◽  
Author(s):  
Lillemor Lewan ◽  
Marianne Andersson ◽  
Paloma Morales-Gomez
Keyword(s):  
Cell Culture ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Cultured Cells ◽  
Sea Water ◽  
Toxicity Testing ◽  
Artemia Salina ◽  
Good Survival ◽  
Cell Culture Medium ◽  
A Cell

This study shows that the Artemia assay, which is usually performed by incubation for a 24-hour period in artificial sea water, can also be performed in phosphate buffered saline (PBS) at pH 7.2, or in a cell culture medium, both of which are used in toxicity assays employing mammalian cells. Thus, by using the equivalent media for incubation, toxic effects in the Artemia assay and toxic mechanisms can more accurately be compared with results obtained in mammalian cell toxicity. Survival of control animals is good for 72 hours, provided that infection can be avoided. A pH greater than 6 is essential for good survival of Artemia Salina, and a pH greater than 10.5 should be avoided. Because of the risk of infection at low saline concentrations, a decreased incubation time of 16 or 12 hours is recommended. The lethal concentrations (LC10 and LC50) in the 24-hour Artemia assay of the first ten chemicals in the MEIC programme were measured in PBS, and the results compared with those from a previous study of the effects in a 10-minute acute ATP leakage assay, specifically indicating lesions in the cell membrane. The EC10 and EC50 values for the alcohols in the Artemia assay were 35–75% of the corresponding values in the ATP leakage assay. For paracetamol and amitriptyline, the EC10 and EC50 values in the Artemia assay were 2–16% of the corresponding values in the ATP-leakage assay. The greatly increased toxicity of the two compounds in the animals may be related to systemic effects. FeSO4 was very toxic to Artemia salina at a concentration of lug/ml, but it did not cause leakage of ATP from cultured cells, even at a concentration of 8,000μg/ml, showing that widely different mechanisms of interaction with FeSO4 are measured by the two assays. A lower toxicity of polygodial, a sesquiterpenoid unsaturated dialdehyde, in cell culture medium, was obvious in the Artemia assay. Thus, some factors in cell culture medium must, by interacting with the toxic molecule, protect the animals against the toxicity of polygodial, as was previously found in cultured cells.

scholarly journals Effects of Zataria multiflora essential oil on the germinative cells of Echinococcus granulosus

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Nasim Kowsari ◽  
Mohammad Moazeni ◽  
Ali Mohammadi
Keyword(s):  
Cell Culture ◽  
Essential Oil ◽  
Echinococcus Granulosus ◽  
Culture Medium ◽  
Cultured Cells ◽  
Lethal Effect ◽  
Trypan Blue Exclusion ◽  
Zataria Multiflora ◽  
Biological Studies

Abstract Background Novel and more efficient compounds are urgently required for medical treatment of cystic echinococcosis (CE). Germinative cell culture of Echinococcus granulosus could be used for anti-echinococcosis agent tests and other biological studies on CE. This study was performed to establish an in vitro cell culture model for E. granulosus germinative cells and to evaluate the lethal effect of Zataria multiflora essential oil (ZMEO) on the cultured cells. Methods The inner surface of germinal layers of CE cysts was scraped, and the obtained materials were trypsinized to obtain a suspension of single germinative cells. Medium 199 was used as the basic culture medium and was supplemented with fetal bovine serum, 2-mercaptoethanol, l-cysteine, l-glutamine, glucose, sodium pyruvate, hydatid fluid, amphotericin B and antibiotics. The cells were cultured at a concentration of 104 cells/ml of culture medium and incubated at 37 °C. The culture medium was replaced every 7 days. Chemical composition of ZMEO was identified by GC-MS analysis. ZMEO was tested at concentrations of 0.5–8 mg/ml. Viability of the cells was assessed by trypan blue exclusion assay. Results A significant increase in the cell number was evident at 20, 30 and 45 days after cultivation. At 45 days of cultivation, the number of cells was approximately five-fold higher than on the first day. In GC-MC analysis, carvacrol, p-cymene, g-terpinene and thymol were found to be the main compounds of ZMEO. The lethal effect of ZMEO on the germinative cells at concentrations of 6, 7 and 8 mg/ml was 100% after 60, 25 and 7 min of exposure, respectively. Conclusions At 45 days of cultivation, the cell concentration was suitable for the desired in vitro experiments. A high lethal effect of ZMEO on the germinative cells of E. granulosus may be considered an opportunity for the introduction of a novel, more effective and safer therapeutic agent for treatment of CE using an herbal product. Graphic abstract

scholarly journals Effects of the Antioxidant α-Lipoic Acid on Human Umbilical Vein Endothelial Cells Infected with Rickettsia rickettsii

1998 ◽  
Vol 66 (5) ◽  
pp. 2290-2299 ◽  
Author(s):  
Marina E. Eremeeva ◽  
David J. Silverman
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Lipoic Acid ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Vero Cells ◽  
Cell Culture Medium ◽  
Rickettsia Rickettsii ◽  
Infected Cells ◽  
Umbilical Vein Endothelial Cells

ABSTRACT Rickettsia rickettsii infection of endothelial cells is manifested in very distinctive changes in cell morphology, consisting of extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope and blebbing of the plasma membrane, as seen by transmission electron microscopy (D. J. Silverman, Infect. Immun. 44:545–553, 1984). These changes in cellular architecture are thought to be due to oxidant-mediated cell injury, since their occurrence correlates with dramatic alterations in cellular metabolism, particularly with regard to antioxidant systems. In this study, it was shown that R. rickettsii infection of human umbilical vein endothelial cells resulted in a significant depletion of intracellular reduced glutathione (thiol) content at 72 and 96 h and decreased glutathione peroxidase activity at 72 h postinfection. Infected cells displayed a dramatic increase in the concentration of intracellular peroxides by 72 h. Supplementation of the cell culture medium with 100, 200, or 500 μM α-lipoic acid, a metabolic antioxidant, after inoculation with R. rickettsii restored the intracellular levels of thiols and glutathione peroxidase and reduced the intracellular peroxide levels in infected cells. These effects were dose dependent. Treated infected monolayers maintained better viability at 96 h after inoculation with R. rickettsii than did untreated infected cells. Moreover, supplementation of the cell culture medium with 100 μM α-lipoic acid for 72 h after infection prevented the occurrence of morphological changes in the infected cells. The presence of 100 or 200 μM α-lipoic acid did not influence rickettsial growth in endothelial cells, nor did it affect the ability of R. rickettsii to form lytic plaques in Vero cells. Treatment with 500 μM α-lipoic acid decreased by 50% both the number and size of lytic plaques in Vero cells, and it also decreased the recovery of viable rickettsiae from endothelial cells. However, under all treatment conditions, a significant number of rickettsiae could be detected microscopically. Furthermore, the rickettsiae apparently retained their capacity for intracellular movement, since they possessed long polymerized actin tails after 72 and 96 h of treatment regardless of the concentration of α-lipoic acid used. Since α-lipoic acid does not seem to exhibit direct antirickettsial activity except with long-term exposure at very high concentrations, the mechanism of its protective activity for endothelial cells infected with rickettsiae may involve complex changes in cellular metabolism that only indirectly affect rickettsiae.

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