Journal: Scientific Reports

Article Title: Control of Adaptive Immune Responses by Staphylococcus aureus through IL-10, PD-L1, and TLR2

doi: 10.1038/srep00606

Figure Lengend Snippet: Secretion of cytokines. Monocytes were pre-stimulated with or without IFN-γ for 16 hours, then stimulated with SAC (0.0075%) overnight. Cytokines were measured by specific ELISAs with supernatants from 5 to 8 donors. Results represented as mean (pink line) of each group in box plots. (a) TNF-α and IL-12. (b) IL-10. (c) Transcriptional Induction of cytokines and chemokines by SAC. Monocytes (5 x 10 6 /well) were stimulated with SAC for 5 hours. Total RNA was isolated and RT-PCR was carried out as described in Materials and Methods. Data were from one donor representing three donors with similar results. (d) Transcriptional induction of cytokines by SAC for indicated times in the absence or presence of IFN-γ.

Article Snippet: Monocytes were cultured in macrophage-serum free medium (Invitrogen, Carlsbad, CA) and seeded into polystyrene tissue culture 24 well-plates (Costar, Cambridge, Mass.) with the following reagents respectively: Staphylococcus aureus cells (SAC; Cowan I strain, heat-inactivated, from Calbiochem Corporation, La Jolla, CA), LTA (lipoteichoic acid from S. aureus and Streptococcus faecalis, Sigma), LPS (lipopolysaccharide from Escherichia coli K235; List Biological Laboratories, Inc., Campbell, CA.), LPS (from Salmonella typhosa, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella abortus equi, 5 μg/ml, Sigma), IFN-γ(Genentech), anti-human IL-10 neutralizing antibody (10 μg/ml, Invitrogen), anti-human TNF-α neutralizing antibody (10 μg/ml, Endogen, Inc., Cambridge, MA), mouse IgG1 and IgG2a (eBioscience), herbimycin A, genistein, Src kinase inhibitor II (Calbiochem).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Control of Adaptive Immune Responses by Staphylococcus aureus through IL-10, PD-L1, and TLR2

doi: 10.1038/srep00606

Figure Lengend Snippet: Monocytes were cultured in medium in the presence of: (a) indicated reagents (IL-10, 20 ng/mL; IFN-γ, 100 ng/ml and SAC) for 40 hours. Data is representive of results with 3 donors. (b) in the presence of SAC with or without anti-IL-10 neutralizing antibodies or anti-TNF-α neutralizing antibodies for 40 hours. Cells were stained, washed and analyzed on flow cytometer for the expression of CD86, HLA-DR, and CD36. Two additional experiments have been done with similar results. (c) Monocytes were cultured in the presence or absence of SAC for 60 hours. After fixation and permeablization cells were stained with indicated antibodies on coverslips and subject to analysis with the confocal microscope equipped with an objective lens of 63x. Overlay: FITC, DAPI, DIC. (d) Monocytes were cultured in medium and pretreated with genistein (G, 74 μM), herbimycin A (H, 1 μg/ml), or tyrphostin (T, 1 μg/ml) for one hour, then stimulated with SAC for 40 hours. Data were pooled data from three donors. (e) Monocytes were pretreated with indicated inhibitors for five hours, then treated with SAC for 40 hours. Cells were stained with indicated antibodies and subject to analysis on flow cytometer. HerbA, herbimycin A; Src KI, Src kinase inhibitor II; GMFI, geometric mean fluorescence intensity. (f) Induction of NF-κB nuclear translocation in human monocytes by S. aureus . (A) Monocytes (25 x 10 6 /well) were stimulated with either LPS, or SAC for 1 hour. Nuclear extracts (NE) were made and assayed according to Materials and Methods. Data were from one donor representing two with similar results. (B) Nuclear extracts were made as (A) and anti-p50 antibody or anti-p65 antibody was added to the DNA binding reactions and assayed according to Materials and Methods.

Article Snippet: Monocytes were cultured in macrophage-serum free medium (Invitrogen, Carlsbad, CA) and seeded into polystyrene tissue culture 24 well-plates (Costar, Cambridge, Mass.) with the following reagents respectively: Staphylococcus aureus cells (SAC; Cowan I strain, heat-inactivated, from Calbiochem Corporation, La Jolla, CA), LTA (lipoteichoic acid from S. aureus and Streptococcus faecalis, Sigma), LPS (lipopolysaccharide from Escherichia coli K235; List Biological Laboratories, Inc., Campbell, CA.), LPS (from Salmonella typhosa, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella abortus equi, 5 μg/ml, Sigma), IFN-γ(Genentech), anti-human IL-10 neutralizing antibody (10 μg/ml, Invitrogen), anti-human TNF-α neutralizing antibody (10 μg/ml, Endogen, Inc., Cambridge, MA), mouse IgG1 and IgG2a (eBioscience), herbimycin A, genistein, Src kinase inhibitor II (Calbiochem).

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing, Microscopy, Fluorescence, Translocation Assay, Binding Assay