rough los e. coli j5 rc mutant  (Millipore)

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Efficiency of LPCAT2 gene silencing in RAW264.7 macrophages with or without LPS infection. ( A ) The melt curve for the LPCAT2 amplicon was obtained by reverse transcribing LPCAT2 mRNA to cDNA and amplifying cDNA with real-time qPCR. Using R/ggplot2 (method = “gam”, formula = y~s (x, bs = “cs”)), the smooth curve was created from pooled data from all LPCAT2 RT-qPCR experiments (≥3 independent experiments). ( B ) A control group of RAW264.7 macrophages were transiently transfected with negative siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( C ) A group of RAW264.7 macrophages was transiently transfected with LPCAT2 siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. ( D ) LPCAT2 gene expression in the control group is compared with that in the silenced LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. **— p < 0.01, ***— p < 0.001, ****— p < 0.0001.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Infection, Amplification, Quantitative RT-PCR, Control, Transfection, Incubation, RNA Extraction, Gene Expression

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Transcriptional effects of silencing LPCAT2 gene in RAW264.7 macrophages. ( A , E , I ) The melt curves for TLR4 , CD14, and MD2 amplicons were obtained by reverse transcribing mRNA to cDNA and amplifying cDNA with real-time qPCR. Using R/ggplot2 (method = “gam”, formula = y~s (x, bs = “cs”)), the smooth curves were created from pooled data from all TLR4 , CD14 , and MD2 RT-qPCR experiments (≥3 independent experiments). ( B , F , J ) A control group of RAW264.7 macrophages were transiently transfected with negative siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( C , G , K ) A group of RAW264.7 macrophages was transiently transfected with LPCAT2 siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. ( D , H , L ) LPCAT2 gene expression in the control group is compared with that in the silenced LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. *— p ≤ 0.05, **— p < 0.01, ***— p < 0.001, ****— p < 0.0001.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Quantitative RT-PCR, Control, Transfection, Infection, Incubation, RNA Extraction, Gene Expression

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Transcriptional effects of overexpressing LPCAT2 on TLR4 and CD14 gene expression in RAW264.7 macrophages. A control group of RAW264.7 macrophages were transiently transfected with either empty vectors (plasmid vectors with no target sequence) or LPCAT2 vectors (plasmid vectors with LPCAT2 sequence) and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( A – C ) Comparison of LPCAT2 , TLR4, and CD14 gene expression in non-infected and LPS-infected cells. ( D – F ) Comparison of LPCAT2, TLR4 , and CD14 gene expression in the control group and overexpressed LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. *— p ≤ 0.05, **— p < 0.01, ***— p < 0.001, ****— p < 0.0001. Data were obtained from at least three independent experiments.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Gene Expression, Control, Transfection, Plasmid Preparation, Sequencing, Infection, Incubation, RNA Extraction, Quantitative RT-PCR, Comparison

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Transcriptional effects of silencing LPCAT2 gene in RAW264.7 macrophages. ( A , E , I , M ). The melt curves for TNF Alpha , IL6 , IFN Beta , and IP10 amplicons were obtained by reverse transcribing mRNA to cDNA and amplifying cDNA with real-time qPCR. Using R/ggplot2 (method = “gam”, formula = y~s (x, bs = “cs”)), the smooth curves were created from pooled data from all TNF Alpha , IL6 , IFN Beta , and IP10 RT-qPCR experiments (≥3 independent experiments). ( B , F , J , N ) A control group of RAW264.7 macrophages were transiently transfected with negative siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( C , G , K , O ) A group of RAW264.7 macrophages was transiently transfected with LPCAT2 siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. ( D , H , L , P ) LPCAT2 gene expression in the control group is compared with that in the silenced LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. *— p ≤ 0.05, **— p < 0.01, ***— p < 0.001, ****— p < 0.0001.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Quantitative RT-PCR, Control, Transfection, Infection, Incubation, RNA Extraction, Gene Expression

Journal: PLoS ONE

Article Title: Effect of Solar Particle Event Radiation and Hindlimb Suspension on Gastrointestinal Tract Bacterial Translocation and Immune Activation

doi: 10.1371/journal.pone.0044329

Figure Lengend Snippet: Terminal ileum obtained 4 days post irradiation and/or 6 days post hindlimb suspension or from control animals was stained for LPS using a mouse mAb specific for E. coli LPS. A) Ileum from a control treated mouse. B) Ileum from a mouse irradiated with 2 Gy of 70 MeV protons. C) Ileum from a mouse subjected to hindlimb suspension. D) Ileum from a mouse treated with hindlimb suspension and irradiated with 2 Gy of protons. Original magnifications 200×. Tissue from 10 animals was analyzed for each condition.

Article Snippet: Tissue was cut into 6 µm sections in a Leica CM1850 cryostat, fixed in acetone (Thermo Fisher), and stained with rabbit anti-mouse Claudin-3 IgG (Thermo Fisher), goat anti-Lipopolysaccharide IgG (US Biologicals), or a murine monoclonal antibody to Escherichia coli (J5) LPS (Acris Antibodies) and detected with biotinylated anti-rabbit, goat, or mouse IgG (Sigma), streptavidin horseradish-peroxidase (Vector Labs), and AEC substrate (Sigma).

Techniques: Irradiation, Staining

Journal: Frontiers in Aging Neuroscience

Article Title: Lipopolysaccharide, Identified Using an Antibody and by PAS Staining, Is Associated With Corpora amylacea and White Matter Injury in Alzheimer's Disease and Aging Brain

doi: 10.3389/fnagi.2021.705594

Figure Lengend Snippet: PAS staining of LPS in Corpora amylacea of AD and aging brains, and PAS staining of purified LPS. In control, PVWM LPS (A1) and PAS (A2) were co-localized (A3) . IN AD PVWM there were more LPS stained vacuoles (B1) and PAS-stained Corpora amylacea (B2) which were mostly co-localized (B3) . Most but not all LPS stained vesicles were PAS positive and therefore were Corpora amylacea (CA). PAS stained both E. coli J5 LPS and E. coli O111:B4 LPS at the higher concentrations (1,000 ng/μl and 500 ng/μl) with much greater staining intensity for E. coli J5 LPS than E. coli O111:B4 LPS at the same concentrations (C,D) . When the LPS concentrations were 250 ng/μl, PAS only stained E. coli J5 LPS but not the E. coli O111:B4 LPS. However, the difference was not significant. When the LPS concentrations were 125 ng/μl, PAS did not stain either E. coli J5 LPS or E. coli O111:B4 LPS. Note: LPS, lipopolysaccharide; PAS, Periodic acid–Schiff, a marker for CA; J5, E. coli J5 LPS; B4, E. coli O111:B4 LPS; AD, Alzheimer's disease; Bar = 50 μm.

Article Snippet: To further confirm that PAS stains LPS, purified LPS from E. coli , serotype O111:B4 (L2630, Sigma-Aldrich, St. Louis, MO, USA) and its mutant form of E. coli , serotype J5 (ALX-581-014-L002, Enzo Biochem) were used for PAS staining.

Techniques: Staining, Purification, Control, Marker