Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Cell Reports

Article Title: Microglial identity and inflammatory responses are controlled by the combined effects of neurons and astrocytes

doi: 10.1016/j.celrep.2021.108882

Figure Lengend Snippet:

Article Snippet: 3 days after microglial seeding, the media was replaced with pHrodoTM Red E. Coli BioParticlesTM conjugate suspension (1:10 dilution in PBS) prepared following manufacturer’s instructions (ThermoFisher) and then incubated in a CO 2 -free incubator at 37°C for 1 h. The cells were then physically dissociated from the culture dishes, stained with Alexa Flour conjugated 647 anti-rat CD11b antibody (Biolegend) and analyzed on a BD AccuriTM C6 flow cytometer (BD biosciences).

Techniques: Plasmid Preparation, Recombinant, Isolation, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Micromachines

Article Title: A Nitrocellulose Paper-Based Multi-Well Plate for Point-of-Care ELISA

doi: 10.3390/mi13122232

Figure Lengend Snippet: The schematic representation of the ( a ) direct ELISA of rabbit IgG and ( b ) the sandwich ELISA of sperm protein SP-10 on the NC paper multi-well plate.

Article Snippet: The sandwich ELISA protocol is described as follows: The sandwich ELISA of SP-10 starts with the addition of 3 μL of the capture antibody solution (mouse anti-ACRV1 monoclonal antibody in CBS solution, cat #11789-MM01, Sino Biological) with a concentration of 100 μg/mL, onto the NC paper well, followed by the overnight incubation in a 4 °C fridge for the sufficient antibody immobilization, Then, after taking the blocked plate out of the fridge, each NC paper well was pipetted with 10 μL of the wash buffer (0.05% Tween20 in TBS, pH 7.2–7.4) and blotted with an absorbent pad, three times, to remove the unbound antibodies, Next, 3 μL of the blocking buffer (2% BSA in wash buffer) was added to each well ( (b-i)), followed by a 10-min incubation and washing step with 10 μL wash buffer, three times, again to remove the unbound blocking reagents ( (b-ii)), Next, 3 μL of the SP-10 protein solution (cat #10227-H08B, Sino Biological) was added to the blocked NC paper well and incubated for 10 min, for the SP-10 protein to be captured by the immobilized capture antibody probe ( (b-iii)).

Techniques: Direct ELISA, Sandwich ELISA

Journal: EMBO Molecular Medicine

Article Title: Molecular and functional properties of human Plasmodium falciparum CSP C‐terminus antibodies

doi: 10.15252/emmm.202317454

Figure Lengend Snippet: A Anti‐NANP 5 and anti‐C‐CSP serum IgG. Circles represent ELISA area under the curve (AUC) values for individual donors ( n = 12). B Ten most frequent Ig heavy and light chain V gene pairs in PfCSP‐reactive memory B cells (MBCs; n = 1,172). C Usage frequency of IGHV3‐21 , IGHV3‐33 and other IGHV genes in PfCSP‐reactive MBCs ( n = 1,172) and plasmablasts (PBs; n = 2,380). D, E Isotype distribution (D) and somatic hypermutation (SHM) count (E) in IGHV3‐21 and IGHV3‐33 genes from MBCs in (B, C). F NANP 10 (left) and C‐CSP (right) binding of FL‐CSP‐reactive mAbs ( n = 177). Circles represent ELISA AUC values for each mAb. G Frequency of NANP 10 and C‐CSP reactive mAbs per donor. The number of tested mAbs is indicated. H, I Cross‐reactivity of C‐CSP (H) and NANP 10 (I) reactive mAbs with the PfCSP N‐junc, NANP 10 and C‐CSP; n indicates the number of mAbs tested. Data information: Red lines in (A and E) indicate mean values. Black horizontal lines in (F), (H) and (I) indicate the threshold for binding. (E). The statistical significance in E was assessed by two‐tailed Mann–Whitney test: ** P = 0.0017. Data in (A) are representative of two independent technical replicates. Data in (F, H and I) are obtained from three independent technical replicates and indicated as mean. Source data are available online for this figure.

Article Snippet: Serum samples serially diluted at an initial dilution of 1:200 in 1% BSA with PBS, or mAbs at an initial concentration of 4 μg/ml (PfCSP ELISA) or 1 μg/ml (polyreactivity ELISA) in 1× PBS were loaded on the plate and incubated at RT for 1.5 h. Antibodies were detected with goat anti‐human IgG secondary antibody coupled to horseradish peroxidase (HRP) (Jackson Immuno Research; Cat No: 109‐035‐098) diluted at 1:1,000 in blocking buffer (1× PBS, 0.05% (v/v); Tween20, 1 mM EDTA) and then detected with 2,2′‐azino‐bis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium (ABTS) substrate (Roche Diagnostics; Cat No: 11112422001) diluted at 1:1,000 in H 2 O 2 .

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Two Tailed Test, MANN-WHITNEY