PBMCs were thawed and stimulated with 1 ng/ml lipoteichoic acid (LTA) from S. aureus (InvivoGen, San Diego, USA), 1 ng/mL lipopolysaccharide (LPS) from E. coli K12 (Invivogen) or 1 μg/ml CpG ODN M362 (InvivoGen) in AIM V (Life Technologies AB, Täby, Sweden) with 20 μM mercaptoethanol (Sigma-Aldrich) at a concentration of 1 × 106/ml and incubated for 24 hours at 37°C. Unfortunately, enough cells were not always available for all stimuli. Cell cultures were then centrifuged, supernatants were collected and cell pellets were lysed in RLT-buffer (Qiagen GmbH, Hilden, Germany) and stored at −70°C for later mRNA analysis. The mean viability of the freeze/thawed PBMCs was 85.2%.
Lipopolysaccharide lps from e coli k12
Manufactured by InvivoGen
Sourced in United States
Lipopolysaccharide (LPS) from E. coli K12 is an endotoxin, a component of the outer membrane of Gram-negative bacteria. It functions as a potent activator of the innate immune system.
Automatically generated - may contain errors
2 protocols using lipopolysaccharide lps from e coli k12
1
PBMC Stimulation and mRNA Analysis
2
Ethical Monocyte and Animal Study Protocols
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The human monocyte study was conducted according to the principles expressed in the Declaration of Helsinki. All blood donors gave a written-informed consent before collection and use of their samples. All blood donations, provided by the Dutch National Institute for Public Health and the Environment (RIVM, Bilthoven; the Netherlands) were specifically donated for primary cell isolation. This research goal was explicitly approved by the accredited Medical Research Ethics Committee (MREC), METC, Noord-Holland in the Netherlands. All blood samples were processed anonymously.
Animal studies complied with the ARRIVE guideline and were approved by the central committee animal studies (CCD, The Hague; the Netherlands) following the procedures of European legislation guidelines (2010/63/EU and law for animal testing (WOD); the Netherlands). The specific experiments performed in this study were approved by the authority for animal welfare (IvD) and the Scientific committee (WTC) of Intravacc (Bilthoven; the Netherlands).
Reagents Used for Cell Incubation Al(OH) 3 is Alhydrogel Ò 2% (Brenntag Biosector a former organization of Croda Denmark, Frederikssund; Denmark). AlPO 4 is AdjuPhos Ò (Brenntag Biosector, Frederikssund; Denmark). Lipopolysaccharide (LPS) from E.coli K12, referred to as LPS, was used as a positive control and was obtained from Invivogen (San Diego, CA; USA).
Animal studies complied with the ARRIVE guideline and were approved by the central committee animal studies (CCD, The Hague; the Netherlands) following the procedures of European legislation guidelines (2010/63/EU and law for animal testing (WOD); the Netherlands). The specific experiments performed in this study were approved by the authority for animal welfare (IvD) and the Scientific committee (WTC) of Intravacc (Bilthoven; the Netherlands).
Reagents Used for Cell Incubation Al(OH) 3 is Alhydrogel Ò 2% (Brenntag Biosector a former organization of Croda Denmark, Frederikssund; Denmark). AlPO 4 is AdjuPhos Ò (Brenntag Biosector, Frederikssund; Denmark). Lipopolysaccharide (LPS) from E.coli K12, referred to as LPS, was used as a positive control and was obtained from Invivogen (San Diego, CA; USA).
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