bethyl a90 111b  (Bethyl)

Journal: ImmunoHorizons

Article Title: Aberrant humoral immune responses and intestinal homeostasis in Cd38 Bst1 double knockout mice

doi: 10.1093/immhor/vlaf029

Figure Lengend Snippet: Tissue distribution of BST-1 and CD38. (A) Expression levels of Cd38 and Bst1 in various organs from WT mice. RNA was prepared from various organs from 3-mo-old male C57BL/6J mice (n = 4). Amounts of transcripts of Cd38 and Bst1 relative to Actb in each tissue were determined using quantitative real-time reverse-transcription polymerase chain reaction assay. The amount in each tissue was normalized with that in the spleen as 1. (B-F) Immunohistochemical detection of BST-1 or CD38 localizations. Cryosections of 4-mo-old male C57BL/6J mice were stained with rabbit anti-BST-1 serum and rat anti-CD38 mAb, and the bound antibodies were visualized with Cy3-donkey anti-rabbit IgG (red) and Alexa 488 donkey anti-rat IgG antibodies (green), respectively. Reactivities of anti-BST-1 and anti-CD38 antibodies are indicated with red and green, respectively. Merged images of red (a) and green (b) are indicated in panel c. (B) The negative control (nc) was no primary antibody; (C, D) the nc was preimmune rabbit serum and rat IgG1 as primary antibodies. White rectangles in panels C, D, and E indicate the area to be magnified in the images below. (D) The bronchi are indicated by arrowheads. (E) The area of the renal tubules is indicated by dotted lines. A broken line indicates a capsule of the kidney. To take the photo of the kidney, a longer exposure time was applied to increase the visibility of faint signals by anti-CD38 reactivities at glomeruli. (F) For the analyses of intestines, tissues from Cd38 KO and Bst1 KO were used as ncs for staining with anti-CD38 mAb and anti-BST-1 serum, respectively. Representative data from 3 independent experiments are shown.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) plates were coated with goat anti-mouse IgM, IgG3, IgG1, IgG2b, IgG2c, and IgA antibodies (SouthernBiotech) at 10 μg/mL in coating buffer (carbonate-bicarbonate, pH 9.6) at room temperature for 1 h. After washing 3 times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (washing buffer), and blocking with 0.1% bovine serum albumin (BSA) in washing buffer for 1 h, a 50 μL aliquot of mouse serum appropriately diluted for each isotype in PBS was applied and incubated for 1 h at room temperature.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemical staining, Staining, Negative Control

Journal: ImmunoHorizons

Article Title: Aberrant humoral immune responses and intestinal homeostasis in Cd38 Bst1 double knockout mice

doi: 10.1093/immhor/vlaf029

Figure Lengend Snippet: Bst1 KO B cell–intrinsic hyperresponsiveness to LPS. (A) Total Ig productions by 4 strains at 4 to 5 mo old (female n = 6) after stimulation with TNP-LPS were determined using ELISA. Bar graphs indicate mean with SD, and all data points are included. Statistical significance was determined using 2-way analysis of variance test, and Tukey’s multiple comparison test was used for post hoc testing. (B) Histological analyses of polyclonal immunoglobulin production in the spleen in TI-1 antigen responses. Cryosections of the spleens from WT and Bst1 KO at day 14 were stained with ALP-labeled goat anti-mouse IgM, IgG2b, and IgG2c, antibodies and visualized with BCIP-NBT (dark blue). Nuclei were stained with methyl green. Representative data from 6 female mice of each genotype. (C) Immunohistochemical quantification of antibody-producing cells in the spleen. The ratio of the area occupied by polyclonal antibody-producing cells visualized with BCIP-NBT in panel B to the area of 1 field of each splenic section stained for IgM, IgG2b, or IgG2c was calculated with ImageJ software. The mean and SD of relative areas occupied by antibody-producing cells from 6 mice are indicated in the bar graph. Gray and pale coral pink bars indicate data of WT and Bst1 KO mice, respectively. (D) Increased polyclonal IgG2b and IgA productions by BM chimera lacking BST-1 in hematopoietic cells. Four combinations of BM chimera, WT (C57BL/6J)→WT, Bst1 KO→WT, WT→ Bst1 KO, and Bst1 KO→ Bst1 KO (6 BM chimeras for each combination), were stimulated with TNP-LPS, and concentrations of polyclonal serum immunoglobulins were determined using ELISA. Statistical significances were determined using 2-way analysis of variance test, and Tukey’s multiple comparison test was used for post hoc testing. Asterisks of IgG2b are Bst1 KO→WT vs. WT→WT and WT→ Bst1 KO. Asterisks of IgG2c are WT→ Bst1 KO vs. WT→WT and Bst1 KO→WT. Asterisks of IgA are Bst1 KO→ Bst1 KO vs. WT→WT. (E) Increased growth responses of Bst1 KO B cells stimulated with LPS. Splenic B cells purified with MACS B cell isolation kit were stimulated with 10 µg/mL LPS or F(ab’) 2 goat anti-µ antibody and cultured for 72 h. Growth of B cells was estimated by MTT assay, and relative absorbances at 590 nm are shown. Bar graphs indicate mean with SD, and all data points are included. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) plates were coated with goat anti-mouse IgM, IgG3, IgG1, IgG2b, IgG2c, and IgA antibodies (SouthernBiotech) at 10 μg/mL in coating buffer (carbonate-bicarbonate, pH 9.6) at room temperature for 1 h. After washing 3 times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (washing buffer), and blocking with 0.1% bovine serum albumin (BSA) in washing buffer for 1 h, a 50 μL aliquot of mouse serum appropriately diluted for each isotype in PBS was applied and incubated for 1 h at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Staining, Labeling, Immunohistochemical staining, Software, Purification, Cell Isolation, Cell Culture, MTT Assay