scholarly journals Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants

2019 ◽  
Vol 14 (7) ◽  
pp. 1800714 ◽  
Author(s):  
Bernhard Sissolak ◽  
Christian Zabik ◽  
Natasa Saric ◽  
Wolfgang Sommeregger ◽  
Karola Vorauer‐Uhl ◽  
...  
Keyword(s):  
Cell Culture ◽  
Protein Content ◽  
Cell Lysis ◽  
Residual Protein ◽  
Bradford Assay ◽  
Culture Supernatants

Free flow electrophoresis allows quick preparation of extracellular vesicles from cell culture supernatants and human plasma

Cytotherapy ◽  
2021 ◽  
Vol 23 (5) ◽  
pp. S118
Author(s):  
S. Staubach ◽  
T. Tertel ◽  
V. Börger ◽  
C. Grätz ◽  
M. Pfaffl ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Plasma ◽  
Extracellular Vesicles ◽  
Free Flow ◽  
Culture Supernatants

scholarly journals The Effects of Maternal Smoking on Pregnancy and Offspring: Possible Role for EGF?

2021 ◽  
Vol 9 ◽  
Author(s):  
Hamed Janbazacyabar ◽  
Marthe van Daal ◽  
Thea Leusink-Muis ◽  
Ingrid van Ark ◽  
Johan Garssen ◽  
...  
Keyword(s):  
Cell Culture ◽  
Immune Responses ◽  
Spleen Cell ◽  
Cigarette Smoke ◽  
Mammary Glands ◽  
Smoke Exposure ◽  
Cigarette Smoke Exposure ◽  
Positive Correlation ◽  
Spleen Cell Culture ◽  
Culture Supernatants

Cigarette smoke exposure during pregnancy and lactation is associated with adverse pregnancy outcomes. Here, we investigated the effects of maternal smoke exposure on pregnancy and offspring immunity and explored whether, epidermal growth factor (EGF), an important growth-promoting factor in human colostrum and milk, might be a possible missing link in maternal smoke exposure and changes in infants’ immune responses. Pregnant BALB/c mice were exposed to either cigarette smoke or air during gestation and lactation, and effects on pulmonary inflammation in dams and immune responses in offspring were examined. Maternal smoke exposure increased airway hyperresponsiveness and accumulation of inflammatory cells in the lungs of pregnant dams compared to non-pregnant dams. The E-cadherin protein expression was reduced in mammary glands of cigarette smoke-exposed pregnant dams. EGF levels were higher in mammary glands and serum of smoke-exposed pregnant dams compared to air-exposed pregnant dams. Offspring from cigarette smoke-exposed dams exhibited elevated levels of IL-17A, MCP-1, IL-22, and IL-13 in anti-CD3 stimulated spleen cell culture supernatants. EGF levels were also increased in serum of offspring from smoke-exposed dams. A positive correlation was observed between serum EGF levels and neutrophil numbers in bronchoalveolar lavage fluid of the dams. Interestingly, IL-17A, MCP-1, IL-22, IL13, and IFN-γ levels in anti-CD3 stimulated spleen cell culture supernatants of male pups also showed a positive correlation with EGF serum levels. In summary, our results reveal that maternal smoke exposure predisposes dams to exacerbated airway inflammation and offspring to exacerbated immune responses and both phenomena are associated with elevated EGF concentrations.

Is LM7 a new human retrovirus or a mycoplasma virion?

1995 ◽  
Vol 1 (2) ◽  
pp. 88-94 ◽  
Author(s):  
P Froussard
Keyword(s):  
Cell Culture ◽  
Reverse Transcription ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Enveloped Virus ◽  
Polymerase Chain ◽  
Serological Data ◽  
Culture Supernatants ◽  
Viral Genomic Rna ◽  
Human Retrovirus

A putative retrovirus called LM7 was recently isolated from a patient with MS. This retrovirus was detected in LM7 and LM711 cultured human leptomeningeal cells. In the present work, nucleic adds from LM711 cell culture supernatants were purified and subjected to avian myeloblastosis viral (AMV) reverse transcriptase and to random polymerase chain reaction (rPCR) in order to characterize the genomic material of LM7 virions. Analysis of reverse transcription products allowed the detection of an approximately 14 kb ribonucleic acid in all LM711 cell culture supernatants. However, sequencing of rPCR-amplified molecules as well as RNA blotting data showed essentially that all tested cells producing LM7 particles were infected with mycoplasma. Moreover, purification of LM7 particles onto a linear sucrose density gradient established that the 14 kb nucleic acid was always associated with the 1.19–1.21 g ml-1 sucrose fractions, which are known to correspond to the buoyant density of mycoplasma. In addition, no viral genomic RNA was detected in the 1.17 g ml-1 sucrose fraction containing the low reverse transcription activity. These results strongly suggest that microscopic images and serological data could be related to mycoplasma and/or to a virion associated with the bacteria. The LM7 particle might be a new and additional enveloped virus able to infect Mycoplasma hyorhinis hosts. Thus, for instance, it would be presumptuous to assert, with our current understanding, that the LM7 virion is one of the causal agents of MS in humans.

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

scholarly journals Sustainable strategies based on glycine–betaine analogue ionic liquids for the recovery of monoclonal antibodies from cell culture supernatants

2019 ◽  
Vol 21 (20) ◽  
pp. 5671-5682 ◽  
Author(s):  
Emanuel V. Capela ◽  
Alexandre E. Santiago ◽  
Ana F. C. S. Rufino ◽  
Ana P. M. Tavares ◽  
Matheus M. Pereira ◽  
...  
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibodies ◽  
Downstream Processing ◽  
Aqueous Biphasic Systems ◽  
Phase Partitioning ◽  
Hybrid Processes ◽  
Three Phase ◽  
Aqueous Biphasic ◽  
Biphasic Systems ◽  
Culture Supernatants

Ionic-liquid-based aqueous biphasic systems, three phase partitioning methods and hybrid processes combined with ultrafiltration are sustainable strategies for the downstream processing of monoclonal antibodies.

A rapid-response fluorescent probe for the sensitive and selective detection of human albumin in plasma and cell culture supernatants

2016 ◽  
Vol 52 (36) ◽  
pp. 6064-6067 ◽  
Author(s):  
Yi-Ru Wang ◽  
Lei Feng ◽  
Liang Xu ◽  
Yan Li ◽  
Dan-Dan Wang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Binding Site ◽  
Fluorescent Probe ◽  
Human Albumin ◽  
Drug Binding ◽  
Rapid Response ◽  
Sensitive Detection ◽  
Selective Detection ◽  
Drug Binding Site ◽  
Culture Supernatants

A rapid-response fluorescent probeACDMwas developed for selective and sensitive detection of human albumin (HA)viabinding on a non-drug binding site.

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