Journal: The Journal of Cell Biology

Article Title: Cocaine-induced release of CXCL10 from pericytes regulates monocyte transmigration into the CNS

doi: 10.1083/jcb.201712011

Figure Lengend Snippet: Engagement of σ-1R is critical for cocaine-induced CXCL10 expression in HBVPs. (A) Comparative expression of σ-1R mRNA levels by RT-PCR analysis in both HBVPs and HBMECs. (B) Representative Western blot of σ-1R and GM1 in the lipid raft fractions (4–6) isolated from confluent HBVPs either unexposed or exposed to cocaine using sucrose gradient ultracentrifugation. (C) Representative fluorescence images of HBVPs transfected with σ-1R–RFP plasmid (red fluorescence) and stained with CT-B conjugates Alexa Fluor 488 specific for ganglioside GM1-lipid raft marker (green fluorescence). Bar, 10 µm. Overlay and magnified (mag) images are shown; bar, 5 µm. (D) Quantification of colocalization of σ-1R and CT-B. Two-tailed Student’s t test. (E) CXCL10 was assayed by ELISA assay in the supernatants collected from HBVPs pretreated with σ-1R inhibitor BD1047 (10 µM) for 1 h, followed by cocaine exposure for an additional 24 h. One-way ANOVA followed by Bonferroni’s post hoc test was used to determine the statistical significance among multiple groups. (F) Representative Western blot of silencing of σ-1R in HBVPs transfected with si–σ-1R. CXCL10 was assayed by ELISA assay in the supernatants collected from HBVPs transfected with si–σ-1R and nonsense si-Con followed by cocaine exposure. One-way ANOVA followed by Bonferroni’s post hoc test was used to determine the statistical significance among multiple groups. All data are presented as means ± SD of three or four individual experiments (biological replicates). *, P

Article Snippet: The slides or coverslips were washed with PBS and incubated with Alexa Fluor 594–conjugated anti-mouse or anti-rabbit, Alexa Fluor 488–conjugated anti-rabbit or anti-mouse, Alexa Fluor 488–conjugated anti-mouse, or Alexa Fluor 594–conjugated anti-rabbit immunoglobulin G (Invitrogen) for 1 h at RT.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Fluorescence, Transfection, Plasmid Preparation, Staining, Marker, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: The MNN2 Gene Knockout Modulates the Antifungal Resistance of Biofilms of Candida glabrata

doi: 10.3390/biom8040130

Figure Lengend Snippet: Confocal laser scanning microscopy image a 48-h-biofilm of Candida glabrata ATCC2001, C. glabrata ΔHT6 and C. glabrata Δ mnn2 . The biofilm images were acquired using a confocal scanning laser microscope (Olympus BX61, Model FluoView 1000). Filters: DAPI (100 mg/L emissions filters BA 430–470) and Concanavalin A, Alexa Fluor 488 conjugate (50 mg/L emissions filters BA 505–605). Images were acquired with the program FV10-ASW 4.2 (Olympus) using a magnification of 100×. Measure bar: 10 µm.

Article Snippet: Then, to stain the α-mannopyranosyl residues of the glycosylated mannoproteins (that is, mannans) of the cells, they were overlayed with 25 μg/mL Concanavalin A-Alexa Fluor 488 conjugate (ThermoFisher Scientific, Bartlesville, OK, USA) in the dark for 10 min at room temperature [ , , ].

Techniques: Confocal Laser Scanning Microscopy, Microscopy

Journal: Particle and Fibre Toxicology

Article Title: The crystal structure of titanium dioxide nanoparticles influences immune activity in vitro and in vivo

doi: 10.1186/s12989-018-0245-5

Figure Lengend Snippet: a . IL-4 production by LN cells. b . IL-5 production by LN cells. Mice were sensitized with OVA alone, OVA + rutile NP, OVA + anatase NP, or OVA + CB, and challenged with OVA. LN cell preparations were made and incubated with Con A for 24 h. N = 6, mean ± SEM is shown. (*) P

Article Snippet: LN cell suspensions were cultured at 106 cells/mL culture medium with 5 μg/mL Concanavalin A (MP Biomedicals, Irvine, CA, USA) in 96-well tissue culture plates (Nunc, Roskilde, Denmark) for 24 h. Spleen cell suspensions were cultured at 106 cells/mL culture medium with 1 mg/mL OVA in 96-well tissue culture plates (Nunc) for 120 h. Culture conditions were 37 °C in a humidified atmosphere containing 5% CO2 .

Techniques: Mouse Assay, Incubation

Journal: Bioscience of Microbiota, Food and Health

Article Title: Anti-stress effect of the Lactobacillus pentosus strain S-PT84 in mice

doi: 10.12938/bmfh.17-003

Figure Lengend Snippet: Effect of oral administration of the L. pentosus strain S-PT84 on stress-induced immune compromisation of splenocytes. Splenocytes were collected from mice that were fed an AIN-93 M diet with or without S-PT84 for 1 week and then restrained. The number of splenocytes (A), splenic NK activity against target YAC-1 cells (effector:target ratio = 80:1, 40:1, and 20:1) (B), and IFN-γ (C) and IL-4 (D) production levels in splenocytes cultured with Con A (2.5 µg/ml) for 24 hr were measured as described in MATERIALS AND METHODS. Data are presented as the mean ± SE of five mice. Different letters ( a , b and c ) indicate significant differences between groups (p

Article Snippet: Macrophages were cultured with lipopolysaccharide (LPS, from Escherichia coli 055; 100 ng/ml; Wako Pure Chemical industries, Osaka, Japan) for 48 hr, and splenocytes were cultured with Concanavalin A (Con A; 2.5 µg/ml, Wako) for 24 hr at 37°C.

Techniques: Mouse Assay, Activity Assay, Cell Culture

Journal: bioRxiv

Article Title: Automated in situ profiling of chromatin modifications resolves cell types and gene regulatory programs

doi: 10.1101/418681

Figure Lengend Snippet: An automated platform for high-throughput in situ profiling of chromatin proteins. ( a ) AutoCUT RUN workflow. (1) Cells or tissue are bound to Concanavalin A-coated beads, permeabilized with digitonin, and incubated with an antibody targeting a chromatin protein. (2) Samples are arrayed in a 96-well plate and (3) processed on a Biomek robot fitted with a 96-well magnetic plate for magnetic separation during washes (α), and an aluminum chiller block (β) routed to a circulating water bath (γ) for temperature control. (4) AutoCUT RUN produces in 2 days up to 96 libraries that are ready to be pooled and sequenced. ( b ) Hierarchically clustered correlation matrix of AutoCUT RUN profiles of histone-H3 modifications that mark active (pink) and repressed (blue) chromatin in H1 (orange) and K562 (purple) cells. Pearson correlations were calculated using the log 2 transformed values of read counts split into 500 bp bins across the genome.

Article Snippet: Concanavalin A-coated magnetic beads (Bangs Laboratories, ca. no. BP531).

Techniques: High Throughput Screening Assay, In Situ, Incubation, Blocking Assay, Transformation Assay

Journal: Journal of Virology

Article Title: Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes

doi: 10.1128/JVI.00756-19

Figure Lengend Snippet: Gag derivatives containing KR substitutions in the MA-HBR do not show promiscuous localization in cells, unlike those with KT changes. (A and B) HeLa cells were transfected with full-length Gag-YFP (A) and delNC/Gag-YFP (B), which contain the WT MA sequence or 25/26KR, 25/26KT, 29/31KR, or 29/31KT. At 14 h posttransfection, cells were stained with Alexa Fluor 594-conjugated concanavalin A (ConA-AF594), fixed with 4% paraformaldehyde in PBS, and analyzed using a fluorescence microscope. Note that subcellular distributions of delNC/Gag-YFP constructs mirror those of the corresponding full-length Gag-YFP constructs for both WT and MA-HBR mutants. Forty-two to 85 cells were analyzed under each condition across 3 independent experiments. The localization patterns determined by epifluorescence microscopy were confirmed by confocal microscopy. (Top) Representative confocal images. The blue arrowhead indicates the Gag-YFP signal in the intracellular compartments (IC). (Bottom) Bar graphs representing percentages of cells showing the indicated patterns of subcellular distribution for each Gag-YFP construct. ConA-AF594 staining was used as a plasma membrane (PM) marker (not shown). (C, top) Images showing an example of Gag-YFP showing PM-specific localization and ConA-AF594 staining of the PM. Green arrowheads indicate overlap of the two signals at the cell surface in a merged image. (Bottom) Images showing an example of Gag-YFP exhibiting PM and IC localization. The blue arrowhead indicates the Gag-YFP signal in the intracellular compartments. (D) HeLa cells were transfected with full-length WT Gag-YFP or 29/31KA Gag-YFP. At 16 h posttransfection, cells were stained with ConA-AF594, fixed with 4% paraformaldehyde in PBS, and analyzed using a fluorescence microscope. A total of 45 to 62 cells in 3 independent experiments were analyzed. The localization patterns determined by epifluorescence microscopy were confirmed by confocal microscopy. (Top) Representative confocal images. The blue arrowhead indicates the Gag-YFP signal in the intracellular compartments. (Bottom) Bar graphs representing percentages of cell populations as described above.

Article Snippet: HeLa cells transfected with plasmids expressing Gag-YFP were incubated for 1 min at room temperature with Alexa Fluor 594-conjugated concanavalin A (ConA-AF594; Invitrogen) for visualization of the plasma membrane cells at 14 to 16 h posttransfection.

Techniques: Transfection, Sequencing, Staining, Fluorescence, Microscopy, Construct, Epifluorescence Microscopy, Confocal Microscopy, Marker

Journal: The Journal of Cell Biology

Article Title: Inducible fluorescent speckle microscopy

doi: 10.1083/jcb.201506128

Figure Lengend Snippet: Principle and proof of inducible speckle imaging. (a) A single transverse mode laser beam is scattered by a diffuser before the microscope objective, creating a pattern composed of speckles with a characteristic size (correlation length) at the diffraction limit. This high-contrast pattern is generated at all planes: at, before, and after the objective’s focal plane. The beam induces a heterogeneous photoswitch pattern across the sample. (b) A PSF volume comprises a large number of potentially tagged slots. In FSM, slots are randomly occupied, leading to faint density fluctuations at the PSF scale. Increased fluctuations can be induced by narrowing the PSF or, conversely, by clustering fluorescent molecules, as does ISI, to emulate PSF-sized slots. (c) Comparison between simulated and observed fluorescence pattern structure after an ISI pulse on a uniform fluorescent layer (concanavalin A–conjugated Alexa Fluor 488) at different bleaching strengths (γ). Bars, 2 µm. (d) ISI on GFP–α-tubulin in a fixed Drosophila S2 mitotic cell. To produce a roughly uniform fluorophore pool, microtubules were depolymerized using colchicine. Bars: 5 µm; (inset) 1 µm.

Article Snippet: Thin fluorescent layers (experiments in and ) were prepared by coating coverslips with Alexa Fluor 488–conjugated concanavalin A (Molecular Probes).

Techniques: Imaging, Microscopy, Generated, Fluorescence

Journal: Experimental Animals

Article Title: Novel phenotype in beagle dogs characterized by skin response to compound 48/80 focusing on skin mast cell degranulation

doi: 10.1538/expanim.15-0004

Figure Lengend Snippet: Illustration of main pathways to skin response via mast cells based on results shown in Fig. 4 . Nonresponder dogs showed skin responses to HCO-60, histamine dihydrochloride, concanavalin A, and A23187 comparable to those of wild-type dogs but no response to compound 48/80; the dysfunctional route in NR dogs would be downstream of the GPCR.

Article Snippet: Chemicals Compound 48/80 was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), HCO-60 was purchased from Nihon Surfactant Kogyo K.K. (Tokyo, Japan), histamine dihydrochloride was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), Concanavalin A was purchased from Seikagaku Corp. (Tokyo, Japan), and A23187 was purchased from Alomone Labs, Ltd.. (Jerusalem, Israel).

Techniques:

Journal: Experimental Animals

Article Title: Novel phenotype in beagle dogs characterized by skin response to compound 48/80 focusing on skin mast cell degranulation

doi: 10.1538/expanim.15-0004

Figure Lengend Snippet: Vascular permeability to various compounds. Compound 48/80 (A), HCO-60 (B), histamine dihydrochloride (C), concanavalin A (D), and A23187 (E) at 0.01–100 mg/ml ID injected at 0.05 ml/site into the shaved thorax of anesthetized wild-type (WT) and nonresponder (NR) dogs; Evans blue (1 mg/kg) dissolved in saline IV injected just prior to ID injections; and 10 min later, ID injection sites collected for determination of pigment leakage. The significance of the differences between WT and NR groups was determined by Student’s t -test. ** P

Article Snippet: Chemicals Compound 48/80 was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), HCO-60 was purchased from Nihon Surfactant Kogyo K.K. (Tokyo, Japan), histamine dihydrochloride was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), Concanavalin A was purchased from Seikagaku Corp. (Tokyo, Japan), and A23187 was purchased from Alomone Labs, Ltd.. (Jerusalem, Israel).

Techniques: Permeability, Injection