pbad mtfp1  (Addgene inc)

Journal: bioRxiv

Article Title: High cRel-expressing germinal center B cells favor precursor plasma cell generation

doi: 10.64898/2026.04.26.720943

Figure Lengend Snippet: WT and cRel mTFP1 mice were immunized, and the generation of GC B cells (B220+CD95+GL7+) and non-GC B cells (B220+CD95-GL7-) was measured. (A) Flow cytometry plot of GC B cells and non-GC B cells proportion in WT mice. (B) Flow cytometry plot of GC B cells and non-GC B cells proportion in cRel mTFP1 mice. (C) Proportion of GC B cells within the total B cell population in WT and cRel mTFP1 mice. The mean proportion of GC B cells is indicated within the bar (WT mean 10.2 and cRel mTFP1 mean 10.6). (D) The total number of GC B cells per lymph node (LN) in WT and cRel mTFP1 mice. The total number of GC B cells is indicated within the bar (WT mean 4.2×10 5 and cRel mTFP1 mean 4.8×10 5 ). (E) Histogram of cRel expression in GC B cells and non-GC B cells in cRel mTFP1 mice. (F) Proportion changes of cRel gMFI (geometric mean fluorescence intensity) in GC B cells and non-GC B cells from total B cells in cRel mTFP1 mice. gMFI represents cRel expression. 26.7 is the mean % increase in cRel expression in GC B cells compared to non-GC B cells. The proportion change of cRel expression is calculated as follows: 100x(X-Y)/Y. X represents the gMFI of GC B cells or non-GC B cells, and Y represents the gMFI of total B cells, which includes both GC B cells and non-GC B cells. (G) Histogram of cRel expression in GC B cells and non-GC B cells in WT mice stained with cRel antibody. (H) Proportion changes of cRel gMFI in GC B cells and non-GC B cells from total B cells in WT mice stained with cRel antibody. 62 is the mean % increase in cRel expression in GC B cells compared to non-GC B cells. (I) Flow cytometry plot of GC B cells (B220+CD19+CD3e-NK1.1-IgD low CD38-GL7+) and MBCs (B220+CD19+CD3e-NK1.1-IgD low CD38+GL7-) in cRel mTFP1 mice. (J) Histogram of cRel expression in GC B cells and MBCs in cRel mTFP1 mice. (K) Proportion changes of cRel gMFI in GC B cells and MBCs from IgD low B cells in cRel mTFP1 mice. gMFI represents cRel expression. 18.7 is the mean % increase in cRel expression in GC B cells compared to MBCs. The proportion change of cRel expression is calculated as follows: 100x(X-Y)/Y. X represents the gMFI of GC B cells or MBCs, and Y represents the gMFI of IgD low B cells, which includes both GC B cells and MBCs. (L) Flow cytometry plot of GC B cells and MBCs in WT mice stained with cRel antibody. (M) Histogram of cRel expression in GC B cells and MBCs in WT mice. (N) Proportion changes of cRel gMFI in GC B cells and MBCs from IgD low B cells in WT mice. gMFI represents cRel expression. 16 is the mean % increase in cRel expression in GC B cells compared to MBCs. One dot represents one mouse. For (C) and (D) , an unpaired Student’s t-test was performed. For (F), (H), (K) , and (N) , a paired Student’s t-test was performed. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Wildtype C57BL/6J (referred here as WT) (Strain #:000664) were either purchased from Jackson Laboratory or breed in house. mTFP1-cRel (cRel mTFP1 ) mouse strain has been described previously ( ).

Techniques: Flow Cytometry, Expressing, Fluorescence, Staining

Journal: bioRxiv

Article Title: High cRel-expressing germinal center B cells favor precursor plasma cell generation

doi: 10.64898/2026.04.26.720943

Figure Lengend Snippet: WT and cRel mTFP1 mice were immunized, and cRel expression was measured in LZ cells (B220+CD95+GL7+CXCR4 low CD86 high ) and DZ cells (B220+CD95+GL7+CXCR4 high CD86 low ). (A) Flow cytometry plot of LZ cells and DZ cells in cRel mTFP1 mice. (B) Histogram of cRel expression in LZ cells and DZ cells in cRel mTFP1 mice. 6.5% is the proportion of LZ cells that show increased cRel expression compared to DZ cells. (C) Proportion changes of cRel gMFI in LZ and DZ cells from GC B cells in cRel mTFP1 mice. gMFI represents cRel expression. 29 is the mean % increase in cRel expression in LZ cells compared to DZ cells. The proportion change of cRel expression is calculated as follows: 100x(X-Y)/Y. X represents the gMFI of LZ cells or DZ cells, and Y represents the gMFI of GC B cells, which includes both LZ and DZ cells. (D) Proportion of cRel high-expressing LZ and DZ cells in cRel mTFP1 mice. 8.4 is the mean % increase in cRel high-expressing cells in LZ cells compared to DZ cells. (E) Flow cytometry plot of LZ cells and DZ cells in WT mice stained with cRel antibody. (F) Histogram of cRel expression in LZ cells and DZ cells in WT mice. 4.7% is the proportion of LZ cells that show increased cRel expression compared to DZ cells. (G) Proportion changes of cRel gMFI in LZ and DZ cells from GC B cells in WT mice. gMFI represents cRel expression. 34 is the mean % increase in cRel expression in LZ cells compared to DZ cells. (H) Proportion of cRel high-expressing LZ and DZ cells in WT mice. 5.2 is the mean % increase in cRel high-expressing cells in LZ cells compared to DZ cells. (I) Frequency of mismatch errors in the JH558 intronic sequence spanning 700 bp in low and high cRel-expressing LZ cells. The proportions of cells with 0-4 (light gray), 5-9 (gray), and more than 10 (black) mismatches are shown in the respective color zone. The number of clones sequenced in low and high cRel LZ cells is 41 and 39, respectively. Fisher’s exact test is used to compare the number of mutations between groups. Three independent experiments were performed, and 2-4 mice were pooled together for one experiment. (J-M) Quantitative measurement of mRNA expression of nfkbia (J) , c-myc (K) , Irf4 (L) , and Ccnd2 (M) by qPCR in low and high cRel LZ cells. Ubiquitin C (UBC) is used as a housekeeping control. For (C), (D), (G) , and (H) , one dot represents one mouse; for (J-M) , one dot represents one replicate and 3-5 mice pooled per replicate. Paired student’s t-test was performed. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Wildtype C57BL/6J (referred here as WT) (Strain #:000664) were either purchased from Jackson Laboratory or breed in house. mTFP1-cRel (cRel mTFP1 ) mouse strain has been described previously ( ).

Techniques: Expressing, Flow Cytometry, Staining, Sequencing, Clone Assay, Ubiquitin Proteomics, Control

Journal: bioRxiv

Article Title: High cRel-expressing germinal center B cells favor precursor plasma cell generation

doi: 10.64898/2026.04.26.720943

Figure Lengend Snippet: (A) Schematic of experimental design of cRel low and high LZ cells isolation by flow sorting. cRel mTFP1 mice were immunized, and cRel low (bottom 25% in the histogram) and high (top 25% in the histogram) LZ cells were flow sorted. (B-E) Quantitative measurement of mRNA expression of nfkbia (B) , c-myc (C) , Irf4 (D) , and Ccnd2 (E) by qPCR in low and high cRel LZ cells. β-actin is used as a housekeeping control. (F-J) Violin plot depicting enrichment of NFκB target genes (F) , and expression of nfkbia (G) , c-myc (H) , Irf4 (I) , and Ccnd2 (J) by scRNASeq in low and high cRel LZ cells. For figure (B-E) , unpaired student’s t-test was performed. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. For figure (F-J) , A Wilcoxon rank-sum test with a Bonferroni correction was performed to assess overall transcriptional changes in scRNA datasets comparing the cRel high LZ cell population with the cRel low LZ cells. NFκB target genes, Nfkbia, Irf4 , and Ccnd2 were significantly elevated in the cRel high LZ cell population.

Article Snippet: Wildtype C57BL/6J (referred here as WT) (Strain #:000664) were either purchased from Jackson Laboratory or breed in house. mTFP1-cRel (cRel mTFP1 ) mouse strain has been described previously ( ).

Techniques: Isolation, Expressing, Control

Journal: bioRxiv

Article Title: High cRel-expressing germinal center B cells favor precursor plasma cell generation

doi: 10.64898/2026.04.26.720943

Figure Lengend Snippet: cRel low and high cRel LZ cells were flow sorted from cRel mTFP1 immunized mice for single-cell RNASeq. (A) UMAP plot projected 5 different cell clusters of all cell transcriptomes, which include cRel low and high LZ cells. These clusters are categorized by gene expression associated with cell cycle stages (G1, S, and G2-M phases) and by precursor MBC (pre-MBC) and precursor PC (pre-PC) populations. (B-I) . UMAP plot showing gene expression of selected genes relevant to pre-PC, Irf4 (B) , Ccnd2 (C) , Bcl6 (D) , and c-myc (E) , and relevant to pre-MBC ccr6 (F) , Hhex (G) , and Bcl2 (H) , and NFκB target genes (I) (see materials and methods section for NFκB target gene list). The expression level is indicated by the scale bar shown on the side of each plot. The scRNA pipeline incorporates natural-log transformation using log1p. (J) Overlay of cRel low and high LZ cells on the UMAP plot. The number of cells sequenced in cRel low and high LZ cells is ∼1164 and ∼851, respectively. (K) Bar graph on the proportion of cells in each cluster in cRel low and high LZ cells. (L) Comparison of pre-PC relevant genes ( c-myc, Irf4 , and Ccnd2 ) between cRel low and high LZ cells in the G1, S-phase, and G2-M, pre-PC, and pre-MBC. The expression level is indicated by the scale bar on the side of each plot, and the proportion of cells expressing these genes is denoted by the circle size on the side of each plot. (M) Comparison of pre-MBC relevant genes ( Ccr6, Hhex , and Bcl2 ) between cRel low and high LZ cells in the G1, S-phase, and G2-M, pre-PC, and pre-MBC. The expression level is indicated by the scale bar on the side of each plot, and the proportion of cells expressing these genes is denoted by the circle size on the side of each plot.

Article Snippet: Wildtype C57BL/6J (referred here as WT) (Strain #:000664) were either purchased from Jackson Laboratory or breed in house. mTFP1-cRel (cRel mTFP1 ) mouse strain has been described previously ( ).

Techniques: Single Cell, RNA sequencing, Gene Expression, Expressing, Transformation Assay, Comparison

Journal: bioRxiv

Article Title: High cRel-expressing germinal center B cells favor precursor plasma cell generation

doi: 10.64898/2026.04.26.720943

Figure Lengend Snippet: WT and cRel mTFP1 mice were immunized, and enrichment of pre-PCs and pre-MBCs was measured in low and high-cRel-expressing LZ cells. (A-B) Flow cytometry plot of pre-PC (B220+GL7+CD95+CXCR4 low CD86 high Bcl6 low IRF4 high CD69+) in cRel low (A) and high (B) LZ cells in cRel mTFP1 mice. 1.9% and 11.6% are the proportions of pre-PC in low and high cRel LZ cells, respectively. (C) Proportion of pre-PC in low and high cRel high-expressing LZ in cRel mTFP1 mice. 10 is the mean % increase of pre-PC population in cRel high-expressing LZ cells compared to cRel low-expressing LZ cells. (D-E) Flow cytometry plot of pre-PCs in cRel low (D) and high (E) LZ cells in WT mice stained with cRel antibody. 1% and 9.9% are the proportions of pre-PC in low and high cRel LZ cells, respectively. (F) Proportion of pre-PC in low and high cRel high-expressing LZ in WT mice. 9 is the mean % increase of pre-PC population in cRel high-expressing LZ cells compared to cRel low-expressing LZ cells. (G-H) Flow cytometry plot of pre-MBCs (B220+GL7+CD95+CXCR4 low CD86 high CCR6+) in cRel low (G) and high (H) LZ cells in cRel mTFP1 mice. 12.5% and 15% are the proportions of pre-MBC in low and high cRel LZ cells, respectively. (I) Proportion of pre-MBC in low and high cRel high-expressing LZ in cRel mTFP1 mice. 2 is the mean % increase of pre-MBC population in cRel high-expressing LZ cells compared to cRel low-expressing LZ cells. (J-K) Flow cytometry plot of pre-MBCs in cRel low (J) and high (K) LZ cells in WT mice stained with cRel antibody. 17.3% and 16.9% are the proportions of pre-MBC in low and high cRel LZ cells, respectively. (L) Proportion of pre-MBC in low and high cRel high-expressing LZ in WT mice. -1 is the mean % decrease of pre-MBC population in cRel high-expressing LZ cells compared to cRel low-expressing LZ cells. One dot represents one mouse. Paired Student’s t-test was performed. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Wildtype C57BL/6J (referred here as WT) (Strain #:000664) were either purchased from Jackson Laboratory or breed in house. mTFP1-cRel (cRel mTFP1 ) mouse strain has been described previously ( ).

Techniques: Expressing, Flow Cytometry, Staining