Journal: bioRxiv

Article Title: The ICF2 gene Zbtb24 specifically regulates the differentiation of B1 cells via promoting heme synthesis

doi: 10.1101/2023.12.23.573176

Figure Lengend Snippet: Mice (10-week old males in A-D ; and 8-week old females in E-G ) were intraperitoneally (i.p.) immunized with NP 19 -OVA emulsified in IFA (1:1, 50 μg/100 μl/mouse) on D0, followed by i.p. rechallenging with NP 19 -OVA/IFA (3:1, 20 μg/100 μl/mouse) on D53 (only in E-G ). Sera were collected on indicated days and NP-specific antibodies were detected by ELISA. Cells in spleens on D14 ( C&D ) or D70 ( G ) were stained with antibodies, in combination with 7-AAD to exclude dead cells, to visualize the percents of CD19 + CD38 low CD95 high germinal center B (GCB) cells or CD19 -/low CD138 high plasma cells (PC) by flow cytometry. A&E , schematic diagrams illustrating the immunization and bleeding schedules of mice. B&F , bar graphs showing the optical density (OD) values of NP-specific antibodies in sera of Cd19 Cre/+ and Zbtb24 B-CKO mice on indicated days. C, representative pseudo-plots showing the percentages of GCB (within gated 7-AAD - CD19 + B cells) and frequencies of dark zone centroblasts (DZ, CXCR4 high CD86 low ) and light zone centrocytes (LZ, CXCR4 low CD86 high ) within gated GCB cells in control Cd19 Cre/+ and Zbtb24 B-CKO mice. D&G , representative pseudo-plots showing the percentages of PCs in spleens of mice on D14 ( D ) or D70 ( G ). Cumulative data on the percentages or absolute numbers of indicated B-cell subsets were shown in the right of C&D or bottom of G . Each symbol represents a single mouse of the indicated genotype, and numbers below horizonal lines indicate P values while those in brackets denote percentages.

Article Snippet: Total or NP-specific IgM, IgG1, IgG2b, IgG2c, IgG3 and IgA levels were detected by using HRP-coupled goat anti-mouse subtype-specific secondary antibodies (SouthernBiotech).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: The ICF2 gene Zbtb24 specifically regulates the differentiation of B1 cells via promoting heme synthesis

doi: 10.1101/2023.12.23.573176

Figure Lengend Snippet: Mice (female, 8 weeks of age) were i.p. immunized with alum-precipitated NP 19 -OVA (NP-OVA/alum, 100 μg/100 μl/mouse) on D0, and rechallenged with NP 19 -OVA in PBS (50 μg/100 μl/mouse) on D70. A , a schematic diagram depicting the experimental setup. B&C , bar graphs showing the optical density (OD) values of NP-specific antibody subtypes against coated NP 25 -BSA (NP 25 , B ) or NP 2 -BSA (NP 2 , C ) in diluted sera of Zbtb24 loxp/loxp (Cont), Cd19 Cre/+ and Zbtb24 B-CKO mice on indicated days. D , bar graphs showing the ratios of relatively high-affinity (NP 2 ) to low-affinity (NP 25 ) NP-specific IgG1 & IgG2c in Cd19 Cre/+ and Zbtb24 B-CKO mice on D70 & D84. Each dot represents a single mouse of the indicated genotype and numbers below horizonal lines indicate P values determined by Mann-Whitney test. UT, untested.

Article Snippet: Total or NP-specific IgM, IgG1, IgG2b, IgG2c, IgG3 and IgA levels were detected by using HRP-coupled goat anti-mouse subtype-specific secondary antibodies (SouthernBiotech).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: The ICF2 gene Zbtb24 specifically regulates the differentiation of B1 cells via promoting heme synthesis

doi: 10.1101/2023.12.23.573176

Figure Lengend Snippet: CD19 + B cells, enriched from Zbtb24 B-CKO or control Cd19 Cre/+ mice (male, 8 weeks of age) by magnetic beads, were mixed with CD4 + T cells (purified from male WT mice) at the ratio of 2:1 before being intravenously (i.v.) injected into the Rag2 -/- recipient mice (5×10 6 B cells plus 2.5×10 6 T cells per mouse). One day later, recipient mice were immunized with NP 19 -OVA emulsified in IFA (NP-OVA/IFA) intraperitoneally (i.p.). Blood was taken at indicated times, and total or NP-specific antibody levels in sera were determined by ELISA. A , a schematic flow-chart of the experiment setup. B&C , bar graphs showing levels of total ( B) or NP-specific ( C) IgM/IgG or IgG1/IgG2c levels in sera of recipient mice at indicated times. E , bar graphs showing the percentages and absolute numbers of CD19 + B cells or CD19 -/low CD138 + plasma cells (PC) in the spleens (SPL) and bone marrows (BM) of Cd19 Cre/+ vs. Zbtb24 B-CKO mice. Representative pseudo-plots showing the gates for each population were shown in D . Each dot represents a single recipient mouse, and numbers below horizontal lines indicate P values. AU, arbitrary units.

Article Snippet: Total or NP-specific IgM, IgG1, IgG2b, IgG2c, IgG3 and IgA levels were detected by using HRP-coupled goat anti-mouse subtype-specific secondary antibodies (SouthernBiotech).

Techniques: Magnetic Beads, Purification, Injection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The ICF2 gene Zbtb24 specifically regulates the differentiation of B1 cells via promoting heme synthesis

doi: 10.1101/2023.12.23.573176

Figure Lengend Snippet: Mice were intraperitoneally immunized with the type II TID-Ag NP-Ficoll (10 μg/200 μl PBS/mouse, A-E ) or type I TID-Ag NP-LPS (20 μg/200 μl PBS/mouse, F-H ) on D0. Blood was taken at indicated times and NP-specific antibodies in sera were determined by ELISA in plates coated with NP 25 -BSA. A&F , bar graphs showing the optical density (OD) values of NP-specific antibodies in diluted sera of Cd19 Cre/+ vs. Zbtb24 B-CKO mice on indicated days post immunization with NP-Ficoll ( A ) or NP-LPS ( F ). B&C , representative pseudo/zebra-plots illustrating the gating strategies for CD19 + CD138 + plasma blasts (PB)/CD19 low CD138 high plasma cells (PC) and percents of NP + cells within gated PB (PB-NP + ) & PC (PC-NP + ) cells in peritoneal cavities (PeC, B ) or spleens (SPL, C ) of mice on D35 post NP-Ficoll immunization. D&E and G&H , bar graphs showing the percentages & absolute numbers of PB & PC cells as well as the NP + cells within gated PB/PC cells (PB-NP + /PC-NP + , respectively) in PeC ( D&G ) and SPL ( E&H ) of mice on D35 post NP-Ficoll ( D&E ) or on D21 post NP-LPS ( G&H ) immunization. Each symbol represents a single mouse of the indicated genotype (female, 10 weeks of age in A-E ; and male, 9 weeks of age in F-H ), and numbers below horizonal lines in bar graphs indicate P values.

Article Snippet: Total or NP-specific IgM, IgG1, IgG2b, IgG2c, IgG3 and IgA levels were detected by using HRP-coupled goat anti-mouse subtype-specific secondary antibodies (SouthernBiotech).

Techniques: Enzyme-linked Immunosorbent Assay