Journal: Journal of Reproduction & Infertility

Article Title: Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory Cytokine Production and Modulation of TLR2, TLR4 and MyD88 Expression in Human Endometrial Cells

doi:

Figure Lengend Snippet: Assessment of TLR2, TLR4 and MyD88 expression by ESCs and WECs. A: Assessment of TLR2, TLR4 and MyD88 transcripts expression by ESCs and WECs using RT-PCR. B: Flow cytometric analysis of TLR2 and TLR4 expression by ESCs. C: Western blot analysis of TLR2 and TLR4 expression by ESCs. In RT-PCR and Western blot analyses, PBMC was used as positive control. Monocyte gate of PBMC served as positive area in flow cytometry. D: Immunofluorescent staining of TLR2 and TLR4 in ESCs. Monocytes and HL60 cells were used as positive cell controls for TLR2 and TLR4 immunofluorescent stainings, respectively. Reagent negative control (NC) slides received isotype- matched preimmune normal serum. Nuclei were counterstained with DAPI. P1-3: Three representative participants 1-3, ESCs1-3: Endometrial stromal cells from three representative participants, PBMC: Peripheral blood mononuclear cells, NAC: No amplification control

Article Snippet: For TLR2 staining, 5×10 5 living cells were incubated with 5 μg/ml of Alexa fluor 488-conjugated anti-TLR2 antibody (BD) diluted in PBS-BSA for 30 min . All incubations were performed on ice.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Flow Cytometry, Staining, Negative Control, Amplification

Journal: Journal of Reproduction & Infertility

Article Title: Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory Cytokine Production and Modulation of TLR2, TLR4 and MyD88 Expression in Human Endometrial Cells

doi:

Figure Lengend Snippet: Effect of LTA on TLR2 and MyD88 gene expression in WECs. WECs were treated with LTA (10000 ng/ml ) for 8 hr and expression of TLR2 and MyD88 genes was evaluated by RT-PCR. Representative TLR2 and MyD88 PCR bands are shown at the bottom of each graph. AU: Arbitrary unit

Article Snippet: For TLR2 staining, 5×10 5 living cells were incubated with 5 μg/ml of Alexa fluor 488-conjugated anti-TLR2 antibody (BD) diluted in PBS-BSA for 30 min . All incubations were performed on ice.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of gene silencing on fHA-mediated IL-6 production in IVD cells . A ) siRNA-mediated knockdown of genes Toll like receptor ( TLR ) 2 , TLR4 , CD44 and RHAMM was confirmed after 30 hours in intervertebral disc (IVD) cells by qRT-PCR ( n = 4). In each case, gene expression was calculated as fold change as compared to untreated cells. The use of a non-specific scrambled siRNA (siRNA (S)), confirmed specificity of gene knockdown. B, C ) Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( n = 4) ( B ) and lipopolysaccharide (LPS)- (25 ng/ml) ( n = 3). ( C ) stimulated IVD cells following gene knockdown of TLR2 or TLR4 respectively, as determined by IL-6 ELISA. ( D ) hyaluronic acid fragment (fHA)-treated (20 μg/ml) IVD cells following gene knockdown as determined by IL-6 ELISA ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.01.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Knockdown, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

doi: 10.1186/ar4274

Figure Lengend Snippet: The effect of TLR2 inhibition on fHA-mediated IL-6 production in IVD cells . Interleukin (IL)-6 protein production by Pam3CSK4- (25 ng/ml) ( A ) and hyaluronic acid fragment (fHA)-treated (20 μg/ml) ( B ) intervertebral disc (IVD) cells following antibody-mediated neutralization of Toll like receptor (TLR)2 activity ( n = 4). IL-6 protein levels are represented as a percentage of those measured for untreated cells. In all cases, analyses were performed in triplicate and values expressed as mean ± S.D. Statistical analysis was performed using the Student's t -test, * P <0.05 as compared to cells treated with non-specific IgG control antibody.

Article Snippet: Cells were then pre-incubated for one hour with either an affinity purified polyclonal rat anti-human TLR2 neutralizing antibody (final concentration 5 μg/ml) (LabForce, Switzerland) or an isotype matched IgG control (Lucerna-Chem, Luzern, Switzerland).

Techniques: Inhibition, Neutralization, Activity Assay, Control