Journal: PLoS Neglected Tropical Diseases

Article Title: Viridot: An automated virus plaque (immunofocus) counter for the measurement of serological neutralizing responses with application to dengue virus

doi: 10.1371/journal.pntd.0006862

Figure Lengend Snippet: (A and B) Images of DENV4 plaques on C6/36 cells (peroxidase-labeled antibody and TrueBlue stain) acquired using the iPhone 7 plus of the bottom and top of the plate. (C) Images of ZIKV plaques on Vero cells on white plates, with images acquired using the Samsung Galaxy S7. (D) Images of ZIKV plaques on Vero cells acquired using the Zeiss Axio Imager.M1 microscope, using bright top lighting. (E) IFN-γ ELISPOT of the T-cell response to Influenza A nucleoprotein peptide 147–155, imaged with the Zeiss Axio Cam Imager.M2 scope with the AxioCam MRc camera, 5x objective lens. (F) Total (left) and DENV4-specific IgG + memory B cells (right). Spots were detected with TRITC-labeled IgG-specific antibody or Qdot-conjugated fluorescent antibody using an ELISPOT based Fluorospot, with images acquired using the ImmunoSpot Analyzer. (G) LCMV plaques on Vero E6 cells resolved using crystal violet and imaged using the Alpha Innotech digital camera with the Computar H6Z0812M motorized zoom lens.

Article Snippet: A well of ZIKV plaques on Vero cells was acquired using the Zeiss Axio Imager.M1 microscope with a 2.5X lens and the AxioCam MRc mounted camera, using bright top lighting.

Techniques: Labeling, Staining, Microscopy, Enzyme-linked Immunospot

Journal: The Journal of Physiology

Article Title: A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated

doi: 10.1113/JP276432

Figure Lengend Snippet: Representative immunoblot and semi‐quantitative analysis for neutrophil‐specific LY6G/C (A) and M2c macrophage‐specific CD163 (B) in uninjured muscle and at 1, 2, 3 and 4 days post‐injury. Data are means (SD). *Significantly different from OVX at the corresponding time point (n = 5 per condition per time point).

Article Snippet: Gene expression list of inflammatory chemokines/cytokines and related receptors Quantitative PCR using TaqMan methodology was also performed to confirm gene expression of the following chemokines and cytokines ( Cxcl1 [Mm04207460_m1], Cxcl5 [Mm00436451_g1], Ccl2 [Mm00441242_m1] and Spp1 [Mm00436767_m1]) as well as leukocyte cell surface markers ( Ly6g [Mm04934123_m1], Cd68 [Mm03047340_m1], Cd206 [Mm00485148_m1], Cd163 [Mm00474091_m1] and Cd11b [Mm00434455_m1]).

Techniques: Western Blot

Journal: The Journal of Physiology

Article Title: A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated

doi: 10.1113/JP276432

Figure Lengend Snippet: TA muscles from OVX mice (top panels) and OVX+E2 mice (bottom panels) were analysed for CD45+ cell populations repartitioned by FACS to identify neutrophils. CD11b+LY6G− (leukocytes such as monocytes and macrophages; upper left quadrant of each panel), CD11b+LY6G+ (neutrophils; upper right quadrant of each panel) and CD11b−LY6G− (e.g. erythrocytes, fibroblasts, adipocytes, endothelial cells; lower left of each panel) are shown in representative FACS analysis dot plots for each time point.

Article Snippet: Gene expression list of inflammatory chemokines/cytokines and related receptors Quantitative PCR using TaqMan methodology was also performed to confirm gene expression of the following chemokines and cytokines ( Cxcl1 [Mm04207460_m1], Cxcl5 [Mm00436451_g1], Ccl2 [Mm00441242_m1] and Spp1 [Mm00436767_m1]) as well as leukocyte cell surface markers ( Ly6g [Mm04934123_m1], Cd68 [Mm03047340_m1], Cd206 [Mm00485148_m1], Cd163 [Mm00474091_m1] and Cd11b [Mm00434455_m1]).

Techniques: Muscles

Journal: The Journal of Physiology

Article Title: A moderate oestradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated

doi: 10.1113/JP276432

Figure Lengend Snippet: Neutrophils (CD11b+LY6G+ cells), M1 macrophages (CD11b+CD68+ cells) or M2a/M2c macrophages (CD11b+CD206+ cells) were sorted by FACS and presented as a percentage of total CD45+ cells in uninjured muscle and at 1, 2, 3 and 4 days post‐injury. Data are means (SD). *Significantly different from OVX at the corresponding time point (n = 5–6 per condition per time point).

Article Snippet: Gene expression list of inflammatory chemokines/cytokines and related receptors Quantitative PCR using TaqMan methodology was also performed to confirm gene expression of the following chemokines and cytokines ( Cxcl1 [Mm04207460_m1], Cxcl5 [Mm00436451_g1], Ccl2 [Mm00441242_m1] and Spp1 [Mm00436767_m1]) as well as leukocyte cell surface markers ( Ly6g [Mm04934123_m1], Cd68 [Mm03047340_m1], Cd206 [Mm00485148_m1], Cd163 [Mm00474091_m1] and Cd11b [Mm00434455_m1]).

Techniques:

Journal: Heliyon

Article Title: DNMT3A-mediated DNA methylation and transcription inhibition of FZD5 suppresses lung carcinogenesis

doi: 10.1016/j.heliyon.2024.e29733

Figure Lengend Snippet: DNMT3A promotes methylation of the FZD5 promoter. A, Expression of FZD5 in normal CCD-19Lu cells and NSCLC cell lines (NCI–H1299 and Calu-3) examined by RT-qPCR (n = 3); B, DNA methylation level in CCD-19Lu cells and NSCLC cell lines (NCI–H1299 and Calu-3) examined by qMSP analysis (n = 3); C, binding relationship between DNMT1/DNMT2/DNMT3A/DNMT3B) and FZD5 promoter in NCI–H1299 and Calu-3 cells examined by ChIP-qPCR assay (n = 3); D-E, mRNA (E) and protein (F) levels of DNMT3A in CCD-19Lu cells and NSCLC cell lines (NCI–H1299 and Calu-3) determined by RT-qPCR and WB analysis, respectively (n = 3); F-G, mRNA expression of DNMT3A (F) and FZD5 (G) in NCI–H1299 and Calu-3 cells after oe-DNMT3A administration determined by RT-qPCR (n = 3); H, transcription activity of FZD5 promoter in NCI–H1299 and Calu-3 cells examined by luciferase assay. Differences are compared by the one-way or two-way ANOVA. * p < 0.05.

Article Snippet: DNMT1 (1:50, 5032, Cell Signaling Technology, Beverly, MA, USA), DNMT3A (1:50, 3598, Cell Signaling Technology), as well as DNMT3B (1:20, NB300-516, Novus Biologicals) antibodies were then incorporated to the lysates and left overnight at 4 °C.

Techniques: Methylation, Expressing, Quantitative RT-PCR, DNA Methylation Assay, Binding Assay, ChIP-qPCR, Activity Assay, Luciferase

Journal: The Journal of Physiology

Article Title: Direct assessment of leukocyte signalling and cytokine secretion reveals exercise intensity‐dependent reductions in anti‐inflammatory cytokine action

doi: 10.1113/jp286228

Figure Lengend Snippet: Figure 1. Experimental design Anti-inflammatory cytokine action – reflected by the ability of IL-10 and IL-6 to activate STAT3 and inhibit TNF-α secretion ex vivo – was assessed in blood leukocytes before and after work-matched acute exercise bouts varying in intensity (above vs. below lactate threshold) or pattern (continuous vs. intermittent). Isolated monocytes were stimulated with LPS+IFNγ or IL-10 to promote a pro- (‘M1-like’) or anti-inflammatory (‘M2-like’) phenotype, respectively. Changes in circulating leukocyte counts and plasma cytokine concentrations were measured to compare systemic responses to anti-inflammatory action at the cellular level. Plasma cytokines and immune cell counts were analysed at each blood sampling time point (pre-exercise, post-exercise, 30 min post-exercise and 90 min post-exercise). Anti-inflammatory cytokine action was analysed at the pre-, post- and 90 min post-exercise time points. Monocytes were isolated from the immediate post-exercise blood sample and stimulated for 24 h.

Article Snippet: Cells were cultured in RPMI supplemented with 5 mm glucose, 10% fetal bovine serum and 20 mm Hepes in a humidified incubator (37°C, 5% CO2) for 24 h. After 24 h, culture supernatants were collected and stored at −80°C for the measurement of secreted TNF-α and cells from one triplicate well were removed for surface staining with M1 (CD80; CAT# 130-123-253, Miltenyi Biotec) and M2 (CD163; CAT# 130-112-290, Miltenyi Biotec) markers.

Techniques: Ex Vivo, Isolation, Clinical Proteomics, Sampling

Journal: The Journal of Physiology

Article Title: Direct assessment of leukocyte signalling and cytokine secretion reveals exercise intensity‐dependent reductions in anti‐inflammatory cytokine action

doi: 10.1113/jp286228

Figure Lengend Snippet: Figure 6. CD80 and CD163 expression on monocytes stimulated for 24 h with LPS+IFNγ (A; ‘M1-like’) or IL-10 (B; ‘M2-like’), respectively Values are mean ± SD. Blue and red symbols indicate male and female participants, respectively. CD80 and CD163 expression is presented as the percentage change in median fluorescence intensity (MFI) relative to time-matched control (unstimulated) monocyte cultures. Data were analysed using linear mixed effect models with Tukey’s post hoc testing for pairwise comparisons. ∗P = 0.0187, #P = 0.0933, $P = 0.0985 versus CTL.

Article Snippet: Cells were cultured in RPMI supplemented with 5 mm glucose, 10% fetal bovine serum and 20 mm Hepes in a humidified incubator (37°C, 5% CO2) for 24 h. After 24 h, culture supernatants were collected and stored at −80°C for the measurement of secreted TNF-α and cells from one triplicate well were removed for surface staining with M1 (CD80; CAT# 130-123-253, Miltenyi Biotec) and M2 (CD163; CAT# 130-112-290, Miltenyi Biotec) markers.

Techniques: Expressing, Fluorescence, Control