viral stock  (BEI Resources)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retinoic acid promotes brain-homing of CD8+ T cells during congenital CMV infection

doi: 10.4049/jimmunol.2400150

Figure Lengend Snippet: (A) WT mice were uninfected or infected with 100 PFU of MCMV-gB at 0–1 days post-birth. Spleen, thymus, small intestine, ILN, MLN and CLN were harvested at 7-DPI. (B) Quantified percentage of CCR9+ α4β7+ CD8+ T cells (N=4 mice each group). (C) Representative FACS plots of CCR9 and α4β7+ expression on CD8+ T cells. Results shown as mean. (D) Mice were infected with MCMV-gB at birth. CLN, MLN and Spleen were collected at 14-DPI. Quantified data for CCR9+ α4β7+ CD8+ between tetramer+ and tetramer- CD8+ T cells. For statistical analysis, a two-way ANOVA followed by Bonferroni test was performed for multiple comparisons. ***, P < 0.001; ****, P < 0.0001. All experiments were performed three times and data presented is a representation of one replicate.

Article Snippet: MCMV-gB viral stocks were propagated in M2–10B4 cells (CRL-1972, ATCC) as previously described [ 12 ].

Techniques: Infection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retinoic acid promotes brain-homing of CD8+ T cells during congenital CMV infection

doi: 10.4049/jimmunol.2400150

Figure Lengend Snippet: (A) WT mice were uninfected or infected with 100 PFU of MCMV-gB at 0–1 days post-birth. Spleens, ILN, MLN and CLN were harvested at 7-DPI. (B) Cells were stained for ALDEFLUOR assay. Absolute number of ALDH+ cells were quantified (n=4 mice per group). (C) Representative FACS histograms showing ALDEFLUOR staining. Cells were gated against DEAB negative population. (D) Quantification of the individual cell populations within the ALDH+ gate in MCMV infected CLN, MLN and Spleen (n=4 mice per group). Results shown as mean ± SD. For statistical analysis, a two-way ANOVA followed by Bonferroni test was performed for multiple comparisons. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All experiments were performed three times and data presented is a representation of one replicate.

Article Snippet: MCMV-gB viral stocks were propagated in M2–10B4 cells (CRL-1972, ATCC) as previously described [ 12 ].

Techniques: Infection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retinoic acid promotes brain-homing of CD8+ T cells during congenital CMV infection

doi: 10.4049/jimmunol.2400150

Figure Lengend Snippet: WT mice were uninfected or infected with MCMV-gB at 0–1 days post-birth. Spleens, MLN and CLN were harvested at 7-DPI. (A-B) Transcripts for Aldh1a2 , Il13, Csf2 and Gapdh were measured by qRT-PCR (N = 6–9 mice). Data represented as mean ± SEM. (C) Cells were stained for dendritic cells and represented as percentage of live/lineage negative population. (D) Percentage of dendritic cells with cDC1 phenotype (N = 5 mice per group). (E) Percentage of dendritic cells with cDC2 phenotype. Data represented as mean. (F) CLN, MLN and Spleen were collected from MCMV infected animals at 7-DPI and stained with ALDEFLUOR. Number of ALDH+ CD11c+ F4/80- cells were quantified based on CD8α or CD11b. Data represented as mean. (G) Cartoon of CD8+ T cell and dendritic cell co-culture experiment. CD8+ T cells were isolated from uninfected neonatal CD45.2+ gBT-I mice. Antigen presenting cells (APCs) were isolated at 7 days post-birth from CD45.1+ congenic mice and pulsed with gB peptide. Some MCMV infected APCs were negatively enriched for CD8α. (H) Quantification of CCR9+ α4β7+ within the CD45.2+ CD8+ T cell population (N = 4–6 mice). Data represented as mean. For statistical analysis, a one-way ANOVA followed by Bonferroni test was performed for multiple comparisons. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. All experiments were performed two times and data presented is a representation of one replicate.

Article Snippet: MCMV-gB viral stocks were propagated in M2–10B4 cells (CRL-1972, ATCC) as previously described [ 12 ].

Techniques: Infection, Quantitative RT-PCR, Staining, Co-Culture Assay, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Retinoic acid promotes brain-homing of CD8+ T cells during congenital CMV infection

doi: 10.4049/jimmunol.2400150

Figure Lengend Snippet: (A) CD8+ T cells were isolated from dnRAR mice, mixed at a 1:1 ratio with WT-gBTI and co-transferred into CD45.1+ congenic host at birth and infected 4 hours later with MCMV-gB. (B) Data of pairwise comparison between the percentage of WT and dnRAR co-transferred CD8+ T cells within the brain at 17-DPI. (C) CD8+ T cells were isolated from dnRAR fl/fl CD4 Cre/+ -gBTI or dnRAR fl/fl CD4 +/+ -gBTI mice, adoptively transferred into CD45.1+ congenic hosts at birth and infected 4 hours later with MCMV-gB. (D) Quantification of CD45.2+ CD8+ T cells in spleen at 12-DPI. (E) Number of CD45.2+ CD8+ T cells in brain quantified at 12-DPI. (F) Quantification and representative staining of CD69+ CD103+ on CD45.2+ CD8+ T cells at 50-DPI. For statistical analysis, an Unpaired two-tailed t-test with Mann-Whitney correction was performed. For statistical analysis, an two-way ANOVA followed by Bonferroni test was performed. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data in B and C are combined from two independent experiments, all other experiments are representative of technical replicates.

Article Snippet: MCMV-gB viral stocks were propagated in M2–10B4 cells (CRL-1972, ATCC) as previously described [ 12 ].

Techniques: Infection, Isolation, Comparison, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Pathogens

Article Title: High-Sensitivity RT-LAMP for Molecular Detection of O’nyong-nyong ( Alphavirus onyong )

doi: 10.3390/pathogens13100892

Figure Lengend Snippet: Multiple alignment overview of the design of primers. ( a ) Multiple alignment scheme of ONNV, SINV, and CHIKV genomes, showing each position of the genomes, whether they are a match, mismatch, or gap relative to the reference genome ONNV Gulu strain. The region of interest for LAMP primer design is shown in red. ( b ) Detailed view from Bioedit of the region of interest. Bars with arrows represent each primer design for RT-LAMP ONNV . The gap between genomes relative to the reference genome ONNV Gulu strain is shown in red. Each subclade of ONNV (reference ) is represented by subclade 1 (●) and subclade 2 (●●).

Article Snippet: The limit of detection of the RT-LAMP ONNV assay was determined using 10-fold serial dilutions of the original viral stock from BEI Resources, which was diluted in PBS and heat-treated (see Materials and Methods, for details) ( a,b).

Techniques:

Journal: Pathogens

Article Title: High-Sensitivity RT-LAMP for Molecular Detection of O’nyong-nyong ( Alphavirus onyong )

doi: 10.3390/pathogens13100892

Figure Lengend Snippet: Optimization of RT-LAMP ONNV conditions. ( a ) Effect of varying concentrations of MgSO 4 on the RT-LAMP ONNV reaction; ( b ) different reaction temperatures to determine the optimal temperature for the RT-LAMP ONNV reaction; ( c ) agarose gel analysis of the impact of different ratios of outer/inner/loop primers on the RT-LAMP ONNV reaction; ( d ) agarose gel analysis for comparison of the results of the RT-LAMP ONNV reaction at two temperatures (64 °C and 66 °C) over different reaction times; ( e ) agarose gel analysis of the effect of varying concentrations of Bst 3.0 polymerase on the RT-LAMP ONNV reaction; ( f ) agarose gel analysis of the impact of different concentrations of betaine on the RT-LAMP ONNV reaction; ( g ) effect of adding betaine and DMSO to reaction result visualization with SYBR Green I. M: Molecular marker—GeneRuler 1 kb Plus DNA Ladder (Invitrogen, Carlsbad, CA, USA); NTC: Negative control; +/−: Positive/negative RT-LAMP ONNV result.

Article Snippet: The limit of detection of the RT-LAMP ONNV assay was determined using 10-fold serial dilutions of the original viral stock from BEI Resources, which was diluted in PBS and heat-treated (see Materials and Methods, for details) ( a,b).

Techniques: Agarose Gel Electrophoresis, Comparison, SYBR Green Assay, Marker, Negative Control

Journal: Pathogens

Article Title: High-Sensitivity RT-LAMP for Molecular Detection of O’nyong-nyong ( Alphavirus onyong )

doi: 10.3390/pathogens13100892

Figure Lengend Snippet: Comparison of the limit of detection of RT-LAMP ONNV between two different reaction times and specificity of RT-LAMP ONNV . ( a ) Limit of detection for a 20 min reaction time. ( b ) Limit of detection for a 30 min reaction time. ( c ) Agarose gel electrophoresis analysis of the specificity of the assay to detect ONNV, when performed with RNA of CHIKV, MAYV, and SINV. M: Molecular marker—GeneRuler 1 kb Plus DNA Ladder (Invitrogen). NTC: Negative control. RNA: ONNV RNA extract.

Article Snippet: The limit of detection of the RT-LAMP ONNV assay was determined using 10-fold serial dilutions of the original viral stock from BEI Resources, which was diluted in PBS and heat-treated (see Materials and Methods, for details) ( a,b).

Techniques: Comparison, Agarose Gel Electrophoresis, Marker, Negative Control

Journal: Pathogens

Article Title: High-Sensitivity RT-LAMP for Molecular Detection of O’nyong-nyong ( Alphavirus onyong )

doi: 10.3390/pathogens13100892

Figure Lengend Snippet: Schematic overview of RT-LAMP ONNV .

Article Snippet: The limit of detection of the RT-LAMP ONNV assay was determined using 10-fold serial dilutions of the original viral stock from BEI Resources, which was diluted in PBS and heat-treated (see Materials and Methods, for details) ( a,b).

Techniques: