igg2c southern biotech fitc  (SouthernBiotech)

Journal: Science Advances

Article Title: B cell adapter for PI 3-kinase (BCAP) coordinates antigen internalization and trafficking through the B cell receptor

doi: 10.1126/sciadv.adp1747

Figure Lengend Snippet: ( A and B ) Serum anti-VLP antibody titers in control and BCAP KO mice immunized with 2 μg of VLPs containing ssRNA measured at 7, 14, 21, 28, and 35 days after immunization. ( C ) Fluorescence-activated cell sorting (FACS) plots of VLP-immunized control and BCAP KO mice splenocytes stained with fluorescent VLPs, showing VLP + cells in the B220 + gate. FSC-W, forward scatter width. ( D ) Proportion and count of spleen VLP + B cells [gated as in (C)] from control and KO mice immunized with 2 μg of VLPs for 35 days. ( E ) B220 + VLP + GC B cells were identified as Fas + CD38 − cells in control and KO mice. ( F ) Proportion and count of spleen VLP-specific GC B cells [gated as in (E)] from control and KO mice. ( G to I ) Splenocytes were gated as B220 + VLP + Fas − CD38 + non-GC B cells, and the frequencies and count of switched memory and IgM memory B cells were determined on the basis of IgM and IgD levels. ( J ) VLP-specific plasma cells of IgG or IgG2c isotype enumerated by ELISpot assay on bone marrow (BM) cells from control or KO mice harvested at day 35 after immunization. All data are from one of the two independent experiments yielding similar conclusions. Points represent individual mice ( n = 7 mice per group) with mean [(A) and (B)] or mean with SEM [(D) to (J)] shown. P values of less than 0.05 are shown: * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001, calculated with two-way analysis of variance (ANOVA) with multiple comparisons test [(A) and (B)] or Mann-Whitney U test [(D) to (J)]. A.U., arbitrary units.

Article Snippet: IgG2c (FITC, catalog no. 1079-02) and anti-mouse IgM biotinylated (catalog no. 1021-08) were purchased from SouthernBiotech.

Techniques: Control, Fluorescence, FACS, Staining, Clinical Proteomics, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: JCI Insight

Article Title: Bank1 modulates the differentiation and molecular profile of key B cell populations in autoimmunity

doi: 10.1172/jci.insight.179417

Figure Lengend Snippet: Serum levels of antibodies were quantified by ELISA in the TLR7.tg6 model (32 weeks of age), and in the IMQ-induced model (20 weeks of age). ( A ) Serum levels of IgG antidsDNA antibodies in optical density (OD) at 1:1,000 dilution. Total mice analyzed: WT ( n = 14), T7 ( n = 16), T7.B1 –/– ( n = 16); and WT ( n = 16), WT + IMQ ( n = 19), B1 –/– ( n = 16), B1 –/– + IMQ ( n = 19). ( B ) Serum levels of anti-Sm antibodies (U/mL) at 1:100 dilutions. Total mice analyzed: WT ( n = 12), T7 ( n = 15), T7.B1 –/– ( n = 16); and WT ( n = 10), WT + IMQ ( n = 14), B1 –/– ( n = 10), B1 –/– + IMQ ( n = 13). ( C ) Serum levels of total IgG (μg/mL) at 1:100,000 dilutions. Total mice analyzed: WT ( n = 25), T7 ( n = 29), T7.B1 –/– ( n = 29); and WT ( n = 10), WT + IMQ ( n = 20), B1 –/– ( n = 10), B1 –/– + IMQ ( n = 22). ( D ) Serum levels of IgG2c (μg/mL) at 1:10,000 dilution. Total mice analyzed: WT ( n = 15), T7 ( n = 15), T7.B1 –/– ( n = 17); and WT ( n = 10), WT + IMQ ( n = 19), B1 –/– ( n = 9), B1 –/– + IMQ ( n = 22). ( E ) Serum levels of IgM in optical density (OD) at 1:5,000 dilution. Total mice analyzed: WT ( n = 9), T7 ( n = 13), T7.B1 –/– ( n = 12); and WT ( n = 7), WT + IMQ ( n = 7), B1 –/– ( n = 8), B1 –/– + IMQ ( n = 8). Each point represents 1 individual mouse. Data are shown as mean ± SEM. Mann-Whitney U test with Welch’s correction was used to test statistical significance.

Article Snippet: Slides were briefly washed in TBS-T and stained with Fluorescein Peanut Aglutinin–FITC (PNA-FITC) (Vector Laboratories); purified anti–mouse CD169 (MOMA-1) (clone 3D6.112, 142401, BioLegend), followed by goat anti–rat Alexa Fluor 555 secondary antibody (A-21434, Invitrogen); recombinant anti–mouse CD4 antibody (clone EPR19514, ab183685, Abcam), followed by goat anti–rabbit Alexa Fluor 633 secondary antibody (A-21070); anti–mouse CXCR4 (clone 2B11, 14-9991-82, Invitrogen), followed by goat anti–rat APC secondary antibody (A10540, Invitrogen) or CXCR4 APC eFluor 780 (clone 2B11, 47-9991-82, Invitrogen); anti–mouse IgG2c FITC goat (1078-02, SouthernBiotech); anti–mouse B220 ef450 (clone RA3-682, 48-0452-82, Invitrogen); anti–mouse CD138 PE (clone 281-2, 561070, BD Biosciences); and anti–mouse T-bet PE (clone 4B10, 12-5825-82, Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Bank1 modulates the differentiation and molecular profile of key B cell populations in autoimmunity

doi: 10.1172/jci.insight.179417

Figure Lengend Snippet: ( A ) Gating strategy to detect ABCs by flow cytometry. Sequential gating including: FSV620 live cells; FSC-W × FSC-H to gate single cells; SSC-A × FSC-A to gate lymphocytes; finally, ABCs were CD19 + CD3 – CD21 – CD23 – T-bet + CD11b + CD11c + cells. ( B ) Frequency of ABCs among CD19 + B cells from the spleens of the TLR7.tg6 model. Total mice analyzed: WT ( n = 20), T7 ( n = 16), T7.B1 –/– ( n = 26). ( C ) Frequency of ABCs among CD19 + B cells from the spleens of the IMQ-induced model. Total mice analyzed: WT ( n = 15), WT + IMQ ( n = 20), B1 –/– ( n = 14), B1 –/– + IMQ ( n = 21). ( D ) Frequency of IgG + cells among ABC population from the spleens of TLR7.tg6 and IMQ-induced models. Total mice analyzed: WT ( n = 13), T7 ( n = 12), T7.B1 –/– ( n = 19); and WT ( n = 6), WT + IMQ ( n = 13), B1 –/– ( n = 4), B1 –/– + IMQ ( n = 14). ( E ) Frequency of IgG2c + cells among ABC population from the spleens of TLR7.tg6 and IMQ-induced models. Total mice analyzed: WT ( n = 11), T7 ( n = 10), T7.B1 –/– ( n = 17); and WT ( n = 10), WT + IMQ ( n = 16), B1 –/– ( n = 6), B1 –/– + IMQ ( n = 17). Each point represents 1 mouse. Data are shown as mean ± SEM. Mann-Whitney U test with Welch’s correction was used to test statistical significance.

Article Snippet: Slides were briefly washed in TBS-T and stained with Fluorescein Peanut Aglutinin–FITC (PNA-FITC) (Vector Laboratories); purified anti–mouse CD169 (MOMA-1) (clone 3D6.112, 142401, BioLegend), followed by goat anti–rat Alexa Fluor 555 secondary antibody (A-21434, Invitrogen); recombinant anti–mouse CD4 antibody (clone EPR19514, ab183685, Abcam), followed by goat anti–rabbit Alexa Fluor 633 secondary antibody (A-21070); anti–mouse CXCR4 (clone 2B11, 14-9991-82, Invitrogen), followed by goat anti–rat APC secondary antibody (A10540, Invitrogen) or CXCR4 APC eFluor 780 (clone 2B11, 47-9991-82, Invitrogen); anti–mouse IgG2c FITC goat (1078-02, SouthernBiotech); anti–mouse B220 ef450 (clone RA3-682, 48-0452-82, Invitrogen); anti–mouse CD138 PE (clone 281-2, 561070, BD Biosciences); and anti–mouse T-bet PE (clone 4B10, 12-5825-82, Invitrogen).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: JCI Insight

Article Title: Bank1 modulates the differentiation and molecular profile of key B cell populations in autoimmunity

doi: 10.1172/jci.insight.179417

Figure Lengend Snippet: ( A ) Frequency of CXCR4 + IgG2c + cells among CD138 + B220 – cells from the spleens of the TLR7.tg6 model. Total mice analyzed: WT ( n = 7), T7 ( n = 12), T7.B1 –/– ( n = 10). ( B ) Representative cryosections of spleens from WT, TLR7.tg6, and TLR7.tg6. Bank1 –/– mice, stained with anti-B220 DAPI, anti-CXCR4 APC, anti-IgG2c FITC, and anti-CD138 PE. The red arrow shows a digital zoom (×2) selected from each image. Scale bar: 100 μm (left panels); 20 μm (right panels). This representative experiment was conducted 3 different times. All images were captured using a Confocal Laser Microscope Zeiss LSM 710. ( C ) Area in μm 2 of CXCR4 + IgG2c + CD138 + cells in the spleens from 32-week-old TLR7.tg6 model. Total mice analyzed: WT ( n = 5), T7 ( n = 5), T7.B1 –/– ( n = 5). ( D ) Frequency of Tefh cells (CD62L – CD44 + PDGL-1 – CXCR4 + ) among CD3 + CD4 + cells from the spleens of 32-week-old TLR7.tg6 model. Total mice analyzed: WT ( n = 11), T7 ( n = 7), T7.B1 –/– ( n = 13). ( E ) Correlation between the percentage of ABCs among CD19 + B cells and the percentage of Tefh among CD4 + T cells, using Pearson correlation coefficient, in WT, TLR7.tg6, and TLR7.tg6. Bank1 –/– mice. Each point represents 1 individual mouse. Data are shown as mean ± SEM. Mann-Whitney U test with Welch’s correction was used to test statistical significance.

Article Snippet: Slides were briefly washed in TBS-T and stained with Fluorescein Peanut Aglutinin–FITC (PNA-FITC) (Vector Laboratories); purified anti–mouse CD169 (MOMA-1) (clone 3D6.112, 142401, BioLegend), followed by goat anti–rat Alexa Fluor 555 secondary antibody (A-21434, Invitrogen); recombinant anti–mouse CD4 antibody (clone EPR19514, ab183685, Abcam), followed by goat anti–rabbit Alexa Fluor 633 secondary antibody (A-21070); anti–mouse CXCR4 (clone 2B11, 14-9991-82, Invitrogen), followed by goat anti–rat APC secondary antibody (A10540, Invitrogen) or CXCR4 APC eFluor 780 (clone 2B11, 47-9991-82, Invitrogen); anti–mouse IgG2c FITC goat (1078-02, SouthernBiotech); anti–mouse B220 ef450 (clone RA3-682, 48-0452-82, Invitrogen); anti–mouse CD138 PE (clone 281-2, 561070, BD Biosciences); and anti–mouse T-bet PE (clone 4B10, 12-5825-82, Invitrogen).

Techniques: Staining, Microscopy, MANN-WHITNEY