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

Article Title: The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses

doi: 10.4049/jimmunol.1800071

Figure Lengend Snippet: 183cGT/GT mice immunized with TD Ag NP-CGG exhibit normal GCs. 183cGT/GT and WT littermates were i.p. immunized with 100 μg of NP(31)-CGG in alum, boosted on day 10, and sacrificed on day 14. Total splenocytes (A, D, E, and K) and mediastinal LN cells (H, I, J, and L) were analyzed by flow cytometry. Spleen sections were analyzed by IF imaging; scale bar, 50 μm (B, C, F, and G). (A and H) Quantification of GC B cell frequency (GL7+ Fas+ of B220+). (B and C) Representative images (B) and quantification (C) of GC area (GL7+ IgD– B220+). (D and I) Quantification of isotype-switched IgG1+ (IgG1+ IgM+ of GL7+ Fas+ B220+) and Ag-specific (NP+ of GL7+ Fas+ B220+) GC B cell frequency. (E and J) Quantification of DZ (CXCR4hi CD86lo) and LZ (CXCR4lo CD86hi) GC B cell frequency (of GL7+ Fas+ B220+). (F and G) Representative images (F) and quantification (G) of GC LZ area (CD35+ IgD2 B220+). (K and L) Quantification of isotype-switched, Ag-specific early memory B cell frequency (IgG1+ CD38+ NP+ of B220+). n = 5, one experiment. Statistical significance was determined by ratio paired t test. All WT versus 183cGT/GT comparisons failed to meet statistical significance (α = 0.05).

Article Snippet: Secondary Abs for detecting IgM, IgG1, IgG2b, IgG2c, IgG3, and IgAwere purchased from SouthernBiotech (no. 1020–05, 1070–05, 1090–05, 1079–05, 1100–05, and 1040–05, respectively). eBioscience ELISA diluent (no. 00–4202-56) was used as blocking buffer, eBioscience TMB substrate (no. 00–4201-56) was used to develop, and 1 M phosphoric acid was used to stop development.

Techniques: Flow Cytometry, Imaging

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

Article Title: The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses

doi: 10.4049/jimmunol.1800071

Figure Lengend Snippet: 183cGT/GT mice immunized with TD Ag NP-CGG trend for reduced serum Ab induction, although affinity maturation remains intact. (A–J) 183cGT/GT and WT littermates were immunized with 100 μg of NP(31)-CGG in alum, and serum was collected on days 0, 7, 14, 21, and 28. ELISA-based quantification of serum total IgM concentration (A) and induction (B), total IgG1 concentration (C) and induction (D), NP-specific IgM titer (E) and induction (F), and NP-specific IgG1 titer (H) and induction (I). Aicda–/– and null (empty well) are shown to demonstrate isotype specificity and assay limit of quantification, respectively (A and C). Affinity maturation was assessed by ratio of high affinity (NP4) to broad (NP20) Ag binding for IgM (G) and IgG1 (J). IgG1 affinity maturation was indeterminate (#) at day 0, as NP(4)-BSA–binding IgG1 levels were below the level of quantification (J). n = 6, one experiment. Statistical significance was determined by ratio paired t test. *p < 0.05.

Article Snippet: Secondary Abs for detecting IgM, IgG1, IgG2b, IgG2c, IgG3, and IgAwere purchased from SouthernBiotech (no. 1020–05, 1070–05, 1090–05, 1079–05, 1100–05, and 1040–05, respectively). eBioscience ELISA diluent (no. 00–4202-56) was used as blocking buffer, eBioscience TMB substrate (no. 00–4201-56) was used to develop, and 1 M phosphoric acid was used to stop development.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Binding Assay

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

Article Title: The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses

doi: 10.4049/jimmunol.1800071

Figure Lengend Snippet: 183c is dispensable for CSR in B cells stimulated ex vivo. (A–F) MACS-sorted murine splenic mature B cells were cultured and monitored by flow cytometry at 72 and 96 h for frequency of surface IgG3+ cells upon LPS (A) and LII (C) stimulation; IgG1+ upon LI (B), aCI (E), and aCII (F) stimulation; and IgA+ upon LTD stimulation (D). n = 14 for LPS, LI, LTD, and aCI (five experiments); n = 4 for LII and aCII (one experiment). Statistical significance was determined by ratio paired t test. All WT versus 183cGT/GT comparisons failed to meet statistical significance (α = 0.05).

Article Snippet: Secondary Abs for detecting IgM, IgG1, IgG2b, IgG2c, IgG3, and IgAwere purchased from SouthernBiotech (no. 1020–05, 1070–05, 1090–05, 1079–05, 1100–05, and 1040–05, respectively). eBioscience ELISA diluent (no. 00–4202-56) was used as blocking buffer, eBioscience TMB substrate (no. 00–4201-56) was used to develop, and 1 M phosphoric acid was used to stop development.

Techniques: Ex Vivo, Cell Culture, Flow Cytometry

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

Article Title: The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses

doi: 10.4049/jimmunol.1800071

Figure Lengend Snippet: SRBC-immunized 183cGT/GT mice exhibit normal, although LZ skewed, GCs. 183cGT/GT and WT littermates were immunized i.p. with 1 × 109 SRBCs, boosted on day 10, and sacrificed on day 14. Total splenocytes were analyzed by flow cytometry. (A and B) Representative flow cytometry plots (A) and quantification (B) of GC B cell frequency (GL7+ Fas+ of B220+). (C) Quantification of Tfh frequency (Bcl6+ PD1+ CXCR5+ of CD4+ TCRβ+ B220-). n = 4, one experiment. (D and E) Representative flow cytometry plots (D) and quantification (E) of isotype-switched IgG1+ GC B cell frequency (IgG1+ of GL7+ Fas+ B220+). (F–H) Representative flow cytometry plots (F), quantification of DZ (CXCR4hi CD86lo) and LZ (CXCR4lo CD86hi) GC B cell frequency (of GL7+ Fas+ B220+) (G), and DZ/LZ ratio (H). (I and J) Representative flow cytometry plots (I) and quantification (J) of plasmablast (CD138+ B220lo) and plasmacyte (CD138+ B220–) frequency. All n = 10, two experiments, except (H). Statistical significance determined by ratio paired t test. *p < 0.05.

Article Snippet: Secondary Abs for detecting IgM, IgG1, IgG2b, IgG2c, IgG3, and IgAwere purchased from SouthernBiotech (no. 1020–05, 1070–05, 1090–05, 1079–05, 1100–05, and 1040–05, respectively). eBioscience ELISA diluent (no. 00–4202-56) was used as blocking buffer, eBioscience TMB substrate (no. 00–4201-56) was used to develop, and 1 M phosphoric acid was used to stop development.

Techniques: Flow Cytometry

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

Article Title: The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses

doi: 10.4049/jimmunol.1800071

Figure Lengend Snippet: Ab secretion in cultured B cells is not significantly impaired by 183c miRNA ablation. MACS-sorted murine splenic mature B cells were cultured, and supernatant was harvested at 96 h for ELISA. (A–E) Concentration of IgM (A), IgG1 (B), IgG3 (C), IgG2b (D), and IgA (E) in culture supernatant upon indicated stimulation protocol. n = 4, one experiment. Statistical significance was determined by ratio paired t test. *p < 0.05, ***p < 0.0005.

Article Snippet: Secondary Abs for detecting IgM, IgG1, IgG2b, IgG2c, IgG3, and IgAwere purchased from SouthernBiotech (no. 1020–05, 1070–05, 1090–05, 1079–05, 1100–05, and 1040–05, respectively). eBioscience ELISA diluent (no. 00–4202-56) was used as blocking buffer, eBioscience TMB substrate (no. 00–4201-56) was used to develop, and 1 M phosphoric acid was used to stop development.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

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

Article Title: The B Cell Activation-Induced miR-183 Cluster Plays a Minimal Role in Canonical Primary Humoral Responses

doi: 10.4049/jimmunol.1800071

Figure Lengend Snippet: 183c miRNAs are not required for humoral response to TI-2 Ag NP-Ficoll. 183cGT/GT, 183c+/+, Mir182–/–, Mir182+/–, and Mir182+/+ mice were i.p. immunized with 100 μg of NP(30)-AECM-Ficoll in sterile PBS and sacrificed on day 5. Total splenocytes were analyzed by ELISpot. Quantification of IgM- (A), IgG1- (B), and IgG3-secreting (C) plasmablasts per 106 cells. (D and E) NP-specific IgM-secreting plasmablast quantification per 106 cells (D) and representative membrane images of wells seeded with 0.5 × 106 cells (E). (F and G) NP-specific IgG-secreting plasmablast quantification per 106 cells (F) and representative membrane images of wells seeded with 0.5 × 106 cells (G). Numbers below images represent spot counts done in CTL ImmunoSpot software (E and G). n = 7 for 183cGT/GT, 183c+/+, and Mir182–/–; n = 4 for Mir182+/+; n = 3 for Mir182+/–; n = 1 for mock immunization (PBS) controls; one experiment. Statistical significance was determined by ratio paired t test. All 183cGT/GT versus 183c+/+, Mir182–/– versus Mir182+/–, Mir182–/– versus Mir182+/+, and Mir182–/– versus pooled Mir182+/–/Mir182+/+ comparisons failed to meet statistical significance (α = 0.05).

Article Snippet: Secondary Abs for detecting IgM, IgG1, IgG2b, IgG2c, IgG3, and IgAwere purchased from SouthernBiotech (no. 1020–05, 1070–05, 1090–05, 1079–05, 1100–05, and 1040–05, respectively). eBioscience ELISA diluent (no. 00–4202-56) was used as blocking buffer, eBioscience TMB substrate (no. 00–4201-56) was used to develop, and 1 M phosphoric acid was used to stop development.

Techniques: Sterility, Enzyme-linked Immunospot, Membrane, Software