Journal: Nature Communications

Article Title: Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis

doi: 10.1038/ncomms15013

Figure Lengend Snippet: Results are the average of three independent measurements±s.d. using the Luminex customised anti-human cytokine Milliplex kit. *Represents statistically significant different ( P <0.05) between uninfected and Chlamydia- infected as determined using two-way ANOVA.

Article Snippet: TNF-α, IL-1β, IL-6, IL-8 and IL-10 were measured using a Millipore customised Milliplex anti-human cytokine kit and data acquired on a Luminex FLEXMAP 3D system (Millipore).

Techniques: Luminex, Infection

Journal: Nature Communications

Article Title: Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis

doi: 10.1038/ncomms15013

Figure Lengend Snippet: Pathways upregulated in human iPSdMs upon C. trachomatis infection as shown by proteomics.

Article Snippet: TNF-α, IL-1β, IL-6, IL-8 and IL-10 were measured using a Millipore customised Milliplex anti-human cytokine kit and data acquired on a Luminex FLEXMAP 3D system (Millipore).

Techniques: Infection, Activation Assay, Coagulation

Journal: Nature Communications

Article Title: Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis

doi: 10.1038/ncomms15013

Figure Lengend Snippet: ( a , c ) Level of GFP-tagged C. trachomatis infection of human iPSdM CRISPR/Cas9 mutants after 48 h. Results are the averages from three independent measurements±s.d. using the Cellomics CellInsight NXT. ( b ) Production of cytokines from the IRF5 −/− mutant. Results are the average from three biological replicates assessed using the Luminex Multiplex and shown as fold change relative to expression of each cytokine in KOLF2 parent iPSdMs. *Represent statistically significant different ( P <0.05) between parent and mutants as determined using two-way ANOVA. Equal numbers of WT and mutant cells were seeded for each experiment.

Article Snippet: TNF-α, IL-1β, IL-6, IL-8 and IL-10 were measured using a Millipore customised Milliplex anti-human cytokine kit and data acquired on a Luminex FLEXMAP 3D system (Millipore).

Techniques: Infection, CRISPR, Mutagenesis, Luminex, Multiplex Assay, Expressing

Journal: eBioMedicine

Article Title: Danger signals released during cold ischemia storage activate NLRP3 inflammasome in myeloid cells and influence early allograft function in liver transplantation

doi: 10.1016/j.ebiom.2022.104419

Figure Lengend Snippet: eiOPS activates NLRP3 inflammasome in human myeloid cells. (a) Release of IL-1β by LPS-primed THP-1 cells after incubation with Celsior® or eiOPS for 4 or 16 h. Incubation for 30 min with potassium ionophore nigericin (10 μM) or ATP (3 mM) were used as positive control for NLRP3 activation. Presence of IL-1β in Celsior® and eiOPS before incubation with THP-1 cells was determined as negative control. n = 5. (b) Release of IL-1β by LPS-primed THP-1 cells after incubation with eiOPS for 16 h in the presence of caspase-1 inhibitor Ac-YVAD-AOM (100 μM) or NLRP3 inhibitor MCC950 (10 μM). n = 8. (c) Release of IL-1β by LPS-primed wild-type (WT), NLRP3 −/− , PYCARD −/− , and CASP1 −/− THP-1 cells after incubation with eiOPS for 16 h n = 6. (d) Representative microscopy images of ASC specks in THP-1 cells as detected by immunofluorescence, and percentage of THP-1 cells with at least one ASC speck. Quantification of intracellular ASC specks was done from 6 random ×20 images/conditions in at least 3 independent experiments. (e) Release of IL-1β by LPS-primed human primary monocytes after incubation with eiOPS for 16 h in the presence of Ac-YVAD-AOM (100 μM) or MCC950 (10 μM). Incubation with nigericin (10 μM) or ATP (3 mM) after LPS priming were used as canonical NLRP3 activation positive control. n = 5. Representative pictures in (d) are from 4 independent experiments. Results are presented as mean ± SEM; each sample was tested in duplicate; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001 [one-way ANOVA followed by Bonferroni test (b, c and e) or unpaired Student's t test (d)].

Article Snippet: IL-1β was measured by an IL-1β Human Instant ELISA kit (Invitrogen, Carlsbad, USA) following the manufacturer's instructions and read in a Synergy Mx plate reader (BioTek, Vermont, USA).

Techniques: Incubation, Positive Control, Activation Assay, Negative Control, Microscopy, Immunofluorescence

Journal: eBioMedicine

Article Title: Danger signals released during cold ischemia storage activate NLRP3 inflammasome in myeloid cells and influence early allograft function in liver transplantation

doi: 10.1016/j.ebiom.2022.104419

Figure Lengend Snippet: Different DAMPs in eiOPS are able to activate NLRP3 inflammasome in human primary monocytes. (a) Correlation matrix between concentration of IL-1β released by LPS-primed THP-1 cells after incubation with eiOPS and concentrations of DAMPs present in OPS recovered after cold ischemia storage of liver n = 6. (b–e) Release of IL-1β by LPS-primed human primary monocytes after incubation with eiOPS for 16 h in the presence of specific P2X7R antagonist A438079 (50 μM) (b), phagocytosis inhibitor cytochalasin D (10 μM) or clathrin-mediated endocytosis inhibitor monodansylcadaverine (MDC, 250 μM) (c), specific scavenger of mitochondrial superoxide mitoTEMPO (500 μM) or anti-oxidant N -acetyl-L-cysteine (NAC, 10 mM) (d), and IL-18BP (100 ng/mL), anti-HMGB1, or mouse IgG isotype control (100 ng/mL) (e). Alternatively, eiOPS previously incubated with proteinase K (1 mg/mL) or uricase (2 U/mL) was used (b). n = 6. Results are presented as mean ± SEM; each sample was tested in duplicate; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001 [one-way ANOVA followed by Bonferroni test].

Article Snippet: IL-1β was measured by an IL-1β Human Instant ELISA kit (Invitrogen, Carlsbad, USA) following the manufacturer's instructions and read in a Synergy Mx plate reader (BioTek, Vermont, USA).

Techniques: Concentration Assay, Incubation

Journal: eBioMedicine

Article Title: Danger signals released during cold ischemia storage activate NLRP3 inflammasome in myeloid cells and influence early allograft function in liver transplantation

doi: 10.1016/j.ebiom.2022.104419

Figure Lengend Snippet: DAMPs in eiOPS prime human primary monocytes. (a) Fold change of expression of IL6, TNFA, IL1B , and NLRP3 from human primary monocytes treated with LPS (500 ng/mL) or eiOPS for 16 h as detected by RT-qPCR. n = 4. (b–d) Representative cropped Western blots of intracellular pro-IL-1β (Santa Cruz Biotechnologies Cat# sc-7884, RRID: AB_2124476 ) and β-actin (Santa Cruz Biotechnologies Cat# Sc-47778 HRP, RRID: AB_2714189 ) from human primary monocytes primed for 16 h with eiOPS from different donors (b); different concentrations of LPS (c); and eiOPS in the presence of cytochalasin D (10 μM), MDC (250 μM), IL-18BP (100 ng/mL), anti-HMGB1 (100 ng/mL), anti-TLR2 (100 ng/mL), and mouse IgG isotype control (100 ng/mL), respectively (d). Alternatively, eiOPS previously incubated with proteinase K (1 mg/mL) or DNase I (100 U/mL) was used, as well as TLR2 activator Pam3csK4 (1 μg/mL) (d). (e) Release of IL-1β from human primary monocytes primed with LPS or eiOPS for 16 h, then activated with nigericin for 2 h. n = 4. (f) Release of IL-1β from human primary monocytes primed for 4 h with LPS or Pam3csK4, then incubated with eiOPS for 16 h. n = 4. Representative images are shown from 3 independent experiments. Results are presented as mean ± SEM; each sample was tested in duplicate; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001 [one-way ANOVA followed by Bonferroni test (a) or unpaired Student's t test (e, f)].

Article Snippet: IL-1β was measured by an IL-1β Human Instant ELISA kit (Invitrogen, Carlsbad, USA) following the manufacturer's instructions and read in a Synergy Mx plate reader (BioTek, Vermont, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation