Journal: Frontiers in Microbiology

Article Title: Combining flagellin and human β-defensin-3 to combat bacterial infections

doi: 10.3389/fmicb.2014.00673

Figure Lengend Snippet: In vitro activity of F and FBD3 proteins-secretion of IL-8. (A,B) FACS analysis of Toll-Like-Receptor-5 on Colo-205 and Bjab cell lines. (C,D) In vitro activity of the F protein on Colo-205 and Bjab cells. Colo-205 or Bjab cells (10 5 cells/ml) were incubated with 20 ng (protein) of F for 4 h. The medium separated from the various samples was tested and the amount of the IL-8 was measured as described in Methods. One representative experiment out of two performed is shown (C) . Variations were ±10%. Colo-205 cells (10 5 cells/ml) were incubated with different concentrations of F (1.2 and 12 ng protein), FBD3 (2 or 20 ng fusion protein; comprising about 1.8 and 18 ng, respectively, of the F protein) and GnRH-Cherry for 4 h (20 ng protein). The medium from the different samples was analyzed and the amount of the IL-8 was measured by an Elisa assay as described in the “Methods.” One representative experiment out of four performed is shown (D) .

Article Snippet: After 4 h incubation the cell-free supernatants were analyzed according to the manufacturer’s instructions for human IL-8 content, using Human IL-8 ELIZA KIT II (BD Biosciences San Diego, CA, USA).

Techniques: In Vitro, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Medical mycology

Article Title: A fucose specific lectin from Aspergillus flavus induced interleukin-8 expression is mediated by mitogen activated protein kinase p38.

doi: 10.1093/mmy/myw066

Figure Lengend Snippet: Figure 3. Interleukin-8 expression profile upon FFL treatment: Cells were treated with indicated concentrations of FFL for different time period. Total RNA was isolated and cDNA was synthesized as mentioned, mRNA expression level was determined by Q-PCR method. (A) Cell viability assay: Cells were treated with varying concentrations of FFL for 24 hours. Cell viability was determined as explained in methods. The untreated cells were considered as 100% viable. (B) Cell number assay: Cells were stimulated with varying concentrations of FFL for 24 hours. The cell number in untreated sample was taken as 100%. Trypan blue exclusion assay was used to count the cells. (C) Time dependent expression of IL-8 mRNA in L-132 cells. Cells without treatment were used as control. (D) Dose dependent expression of IL-8 mRNA in L-132 cells. (E) Inhibition of IL-8 mRNA expression by L-fucose: The 40 ng/ml of FFL was preincubated with different concentrations of L-fucose (0.1 mM, 0.2 mM, and 0.3 mM) for 1 hour prior treatment to the cells for indicated time. RNA was isolated and mRNA expression level was determined as explained earlier. (F, G) Expression of IL-8 mRNA in L-132, U-937, and PBMCs: Cells were treated with FFL for 6 and 12 hour. The mRNA expression was determined as explained earlier. (H) Measurements of IL-8 production by sandwich ELISA in L-132, U-937, and PBMCs: Cells were treated with 40 ng/ml of FFL for indicated time. After each time course supernatant was collected and IL-8 concentration was determined as mentioned in kit. The cells treated with 100 nM TPA for 4 hours was used as positive control and untreated cells considered as negative control. The data are presented as mean ± SD of three individual experiments that gave similar results. ∗∗P < .01, ∗∗∗P < .001 versus control. #P < .05, ##P < .01, ###P < .001 versus FFL (40 ng/ml).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) Interleukin-8 concentration in cell culture supernatant was determined by sandwich ELISA using human IL-8 ELISA kit from Elabscience, Beijing (E-EL-H0048), following manufacturers protocol.

Techniques: Expressing, Isolation, Synthesized, Viability Assay, Trypan Blue Exclusion Assay, Control, Inhibition, Sandwich ELISA, Concentration Assay, Positive Control, Negative Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Neutrophils from chronic lymphocytic leukemia patients exhibit an increased capacity to release extracellular traps (NETs)

doi: 10.1007/s00262-016-1921-7

Figure Lengend Snippet: IL-8 depletion impairs the priming activity of CLL plasma to release NETs. a IL-8 levels in plasma before (whole plasma) and after depletion with a specific Ab (n = 6). Significance was determined using Wilcoxon matched-pairs test. b Neutrophils from HD were incubated for 40 min with CLL plasma (IL-8 depleted or not), washed twice and stimulated with PMA for 4 h. Elastase activity and DNA concentration were quantified in supernatants (mean ± SEM, n = 10, Friedman test followed by Dunn’s multiple comparison posttest). c Correlation between IL-8 levels in plasma from CLL patients and the capacity of PMA-activated neutrophils to release NETs. Spearman’s correlation coefficients are shown. d Addition of recombinant IL-8 (0.15 ng/ml) to HD plasma primes neutrophils to release NETs (mean ± SEM, n = 5)

Article Snippet: TNFα was from R&D Systems (Minneapolis, MN), PE-anti-human CD69, FITC-anti-human CD80, and human IL-8 ELISA Kit were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Activity Assay, Incubation, Concentration Assay, Comparison, Recombinant

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Neutrophils from chronic lymphocytic leukemia patients exhibit an increased capacity to release extracellular traps (NETs)

doi: 10.1007/s00262-016-1921-7

Figure Lengend Snippet: NETs delay spontaneous apoptosis and increase the expression of activation markers in CLL-B cells. a Shown are representative dot plots and the percentage of Annexin-V-positive cells after 48 h with or without ionomycin-induced NETs. Significance was determined using Wilcoxon matched-pairs test, n = 13. b Expression of activation markers in CLL cells incubated with PMA-induced NETs, supernatants from PFA-fixed neutrophils or medium (mean ± SEM, n = 6–9 CLL). Significance was determined using Friedman test followed by Dunn’s multiple comparison posttest. c Apoptosis and activation markers in CLL cells incubated with medium, supernatants from unstimulated neutrophils or NETs induced by IL-8 + LPS (mean ± SEM, n = 6)

Article Snippet: TNFα was from R&D Systems (Minneapolis, MN), PE-anti-human CD69, FITC-anti-human CD80, and human IL-8 ELISA Kit were purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Expressing, Activation Assay, Incubation, Comparison

Journal: Frontiers in Microbiology

Article Title: Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

doi: 10.3389/fmicb.2019.00879

Figure Lengend Snippet: Production of C. difficile spores, toxins, and host responses to infection. (A) Colony counts of spores recovered after heat treatment, and total cells in the cell-associated C. difficile fraction (infected cell lysates) in the 2D epithelial model. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05 as determined by two-way ANOVA. (B) ELISA for C. difficile toxins A and B shows increased toxin production after extended infection. Toxins were measured from medium obtained from the apical compartment containing uninfected cell layers incubated for 24 or 48 h (Control) or cells infected with C. difficile for 3, 6, 24, or 48 h. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05 as determined by two-way ANOVA. Gray line represents the sensitivity of the test at 0.5 ng/ml. (C) ELISA for human IL-8 indicates increased IL-8 production at 24 and 48 h p.i. IL-8 was measured in medium obtained from the basolateral compartment containing uninfected cell layers incubated for 3, 6, 24, or 48 h (Control) or cells infected with C. difficile for 3–48 h. Gray line represents the limit of detection at 32 pg/ml. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001 as determined by one-way ANOVA with Tukey’s test for multiple comparison.

Article Snippet: IL-8 production was also determined by analysis of basolateral supernatants from the VDC using a human IL-8 ELISA kit (R&D systems, Minneapolis, MN, United States) following the manufacturer’s instruction.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Control, Comparison

Journal: Frontiers in Microbiology

Article Title: Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

doi: 10.3389/fmicb.2019.00879

Figure Lengend Snippet: C. difficile spores and toxin production, and host response to infection in the 3D-VDC model. (A) Colony counts of spores and total cells in the host cell-associated C. difficile fraction (infected cell lysates). Data shown are the mean of three independent experiments and error bars indicate SD, ns, not significant as determined by two-way ANOVA. (B) Toxin A and B levels from apical compartment supernatants as determined by ELISA in the 3D model. Data shown are the mean of three independent experiments and error bars indicate SD, ns, not significant as determined by two-way ANOVA. Gray line represents the sensitivity of the test at 0.5 ng/ml. (C) Human IL-8 levels in supernatants from the basolateral compartments with uninfected cells incubated for 24 h or with cells infected with C. difficile for 3 and 24 h, as determined by ELISA. Gray line represents the limit of detection at 32 pg/ml. Data shown are the mean of three independent experiments and error bars indicate SD, ** p < 0.01, as determined by the one-way ANOVA with Tukey’s test for multiple comparison.

Article Snippet: IL-8 production was also determined by analysis of basolateral supernatants from the VDC using a human IL-8 ELISA kit (R&D systems, Minneapolis, MN, United States) following the manufacturer’s instruction.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Comparison