easysep mouse cd11c positive selection kit ii  (STEMCELL Technologies Inc)

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: In vivo depletion of dendritic cells ablates Peyer's patch B cell activation following rotavirus infection. Mice expressing the diphtheria toxin receptor under control of the CD11c promoter were administered water (With DC) or diphtheria toxin (Depleted DC). Mice were inoculated with PBS (black bar) or 10 3 ID 50 EC wt rotavirus (white bar). Four days following rotavirus exposure, the Peyer's patches were harvested from each animal. ( A ) Peyer's patches from each animal were assessed for activated B cells by flow cytometric detection of CD19 and CD69 or cultured ex vivo in media for 2 days. Each bar represents the mean number of activated cells (CD19 + /CD69 + ) out of the total indicated cell population (CD19 + ; percent activated) from six to seven individual mice ± SD. *, P < 0.05 compared to mice receiving PBS by Mann-Whitney U. #, P < 0.05 compared to mice receiving rotavirus and water by Mann-Whitney U. ( B ) Peyer's patches were cultured ex vivo in media for 2 days followed by quantification of the amount of secreted total rotavirus-specific antibody (IgM, IgG, and IgA) measured by ELISA. Bars indicated mean level of rotavirus-specific antibody as measured by spectrophotometer of n = 5 individual mice. *, P < 0.05 compared to mice receiving PBS by Mann-Whitney U. Two 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: In Vivo, Activation Assay, Infection, Expressing, Control, Cell Culture, Ex Vivo, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Spectrophotometry

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

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: MyD88 expression in CD11c + cells is required for B cell activation but not for interferon alpha induction. ( A ) Cell suspensions from MyD88 −/− mice or the respective WT control mice were treated with rotavirus or media control. Cell suspensions were either untouched (Unsorted); B cells were negatively selected (B cells or B) or co-cultured with positively selected WT or MyD88 −/− dendritic cells (DC). Percent activated B cells (CD19 + /CD69 + ) out of total B cells (CD19 + ) were assessed. Each bar represents the mean of values obtained from three 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 WT cells. ( B ) Splenic cells from the indicated knockout mice were stimulated overnight with media (black), 0.01 µg/µL purified rotavirus (RV, white), boiled rotavirus (dark gray), or 0.01 µg/µL LPS (light gray), and the percent of CD19 + cells that expressed CD69 out of the total CD19 + population was determined by flow cytometry for each mouse. Mean ± SD ( n = 3–7) are shown. *, P < 0.05 compared to rotavirus-treated WT mouse. ( C ) Splenic single cell suspensions from wild type (WT) or MyD88 −/− (MyD88) mice were treated with either media (black bars), rotavirus (white bars), or boiled rotavirus (grey bars) for 24 hours followed by quantitation of interferon alpha in the supernatant by ELISA. Each bar represents the mean amount of interferon alpha obtained from six individual mice ± SD. *, P < 0.05 by Mann-Whitney U compared to media-treated cells. At least 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: Expressing, Activation Assay, Control, Cell Culture, MANN-WHITNEY, Knock-Out, Purification, Flow Cytometry, Quantitation Assay, Enzyme-linked Immunosorbent Assay