Journal: Journal of Virology

Article Title: Dendritic cell expression of MyD88 is required for rotavirus-induced B cell activation

doi: 10.1128/jvi.00653-25

Figure Lengend Snippet: Rotavirus infection activates both B cells and dendritic cells in the murine Peyer's patch. CD-1 mice were orally inoculated with PBS (black bars) or 10 5 ID 50 EC wt (white bars), and single cell suspensions were prepared from the Peyer's patches 48 hours later. Cells were incubated with antibodies against the indicated cell surface markers and activation marker CD69, and fluorescently labeled cells were detected and quantified using a flow cytometer. ( A, B ) Representative histograms indicating CD69 staining in total Peyer's patch cells isolated from uninfected ( A ) and infected ( B ) animals. (C) Quantification of CD69 staining in each indicated specific cell population. Each bar represents the mean number of activated cells (CD69 + ) out of the total indicated cell population (CD19, CD138, CD11b, CD11c, CD4, or CD8; percent activated) from three individual mice ± SD. *, P < 0.05 compared to PBS-treated animals by Mann-Whitney U. Three independent replicate experiments were performed, and a representative experiment is shown.

Article Snippet: CD90 + , CD11c + , CD11b + , and CD4 + cells were positively selected using the EasySep Mouse CD11c Positive Selection Kit, the EasySep Mouse CD11b Positive Selection Kit, and the EasySep Mouse CD4 Positive Selection Kit (StemCell Technologies, Vancouver, BC, Canada) and the Miltenyi pan DC microbeads or the Miltenyi CD90 microbeads (Miltenyi Biotec, Inc., Auburn, CA), respectively, following the manufacturer's instructions.

Techniques: Infection, Incubation, Activation Assay, Marker, Labeling, Flow Cytometry, Staining, Isolation, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Dendritic cell expression of MyD88 is required for rotavirus-induced B cell activation

doi: 10.1128/jvi.00653-25

Figure Lengend Snippet: CD11c + dendritic cells, but not CD90 + or CD11b + , are required for rotavirus-induced B cell activation. ( A ) Representative flow cytometry dot plots of rotavirus-treated and untreated splenic single cell suspensions isolated from CD-1 outbred mice prior to (unsorted) or following sorting (depleted). Cells were treated with rotavirus (white) or media only (black). B cell activation was assessed by expression of CD69 on the CD19 + population using fluorescently labeled antibodies and flow cytometry, and data are shown as mean percentage of activated B cells out of total B cells ( n = 3) ± SD. ( B ) Undepleted splenic cell suspensions or cell suspensions depleted of all non-B cells (purified B), dendritic cells (CD11c + depleted), T cells (CD90 + depleted), or macrophages (CD11b + depleted) were treated with media (black) or rotavirus (white), and activated B cells (CD19 + /CD69 + ) out of total B cells (CD19 + ) were quantified by flow cytometry. Each bar represents the mean number of activated B cells out of total B cells ( n = 3 individual mice) + SD. *, P < 0.05 by Mann-Whitney U compared to media-treated cells. #, P < 0.05 by Mann-Whitney U compared to presort rotavirus-treated cells. ( C ) Splenic single cell suspensions were treated prior to selection (Unsorted) or depleted of CD11c+ (Sorted) cells prior to treatment. CD11c + cells that were depleted from the cell suspensions were added back prior to treatment (Add Back). Cells suspensions were treated with media (black bar), rotavirus (white bars), or lipopolysaccharide (gray bars). Each bar represents the mean number of activated B cells out of total B cells ( n = 3 individual mice) + SD. *, P < 0.05 by Mann-Whitney U compared to media-treated cells. #, P < 0.05 by Mann-Whitney U compared to unsorted rotavirus-treated cells. ( D ) Splenic single cell suspensions were stimulated with media (black bar) or rotavirus (white bar) prior to selection (Unsorted), following selection (Purified B), or after co-culturing with either CD4 + T cells (T + B) or with the Cd11c + cell population (DC + B). Each bar represents the mean number of activated B cells out of total B cells ( n = 3 individual mice) + SD. *, P < 0.05 by Mann-Whitney U compared to media-treated cells. #, P < 0.05 by Mann-Whitney U compared to unsorted rotavirus-treated cells. Three independent replicate experiments were performed, and a representative experiment is shown.

Article Snippet: CD90 + , CD11c + , CD11b + , and CD4 + cells were positively selected using the EasySep Mouse CD11c Positive Selection Kit, the EasySep Mouse CD11b Positive Selection Kit, and the EasySep Mouse CD4 Positive Selection Kit (StemCell Technologies, Vancouver, BC, Canada) and the Miltenyi pan DC microbeads or the Miltenyi CD90 microbeads (Miltenyi Biotec, Inc., Auburn, CA), respectively, following the manufacturer's instructions.

Techniques: Activation Assay, Flow Cytometry, Isolation, Expressing, Labeling, Purification, MANN-WHITNEY, Selection