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R&D Systems recombinant cd40l protein
Recombinant Cd40l Protein, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+mouse+cd40+ligand/Recombinant+Mouse+CD40+Ligand%2FTNFSF5+(HA-tag)+Protein/pm42001092-134-44-47
Average 94 stars, based on 15 article reviews

recombinant cd40l protein - by Bioz Stars, 2026-09

94/100 stars

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Cell Culture:

Article Title: CD40 promotes MHC class II expression on adipose tissue macrophages and regulates adipose tissue CD4 + T cells with obesity
Article Snippet: .. Adherent cells were cultured overnight before being treated with recombinant mouse CD40 ligand (CD40L; R&D Systems, Minneapolis, MN, USA) for 6 h. Flow cytometry analysis Blood leukocytes were prepared as described [ 34 ]. .. Single-cell suspensions were prepared from spleen and thymus by pressing tissues through a 40-μm cell strainer (BD Falcon; BD Biosciences, Franklin Lakes, NJ, USA).

Article Title: T Follicular Helper Cell-Germinal Center B Cell Interaction Strength Regulates Entry into Plasma Cell or Recycling Germinal Center Cell Fate.
Article Snippet: .. In vitro culture Sorted LZ GC B cells (5000-10,000 cells) were cultured in 96-well U-bottom plates with recombinant mouse CD40 ligand (100 ng/ml, R&DSystems) or recombinantmouse IL-21 (10 ng/ml, R&DSystems) for 5 h. For the analysis of antigen-inducedGCBcell responses, sorted LZ GC B cells (5000-10,000 cells) or LZ Fraction 1-3 cells (2000-5000 cells) were plated into 96-well V-bottom plate and then incubated with 1 or 10 mMOVA323-339 for 2 h at 37 C. Sorted CXCR5+ OT-II CD4+ T cells (10,000 cells) were added to each well and centrifuged briefly. .. In the conjugate formation assay, GC B cells and OT-II T cells were incubated for 30-60 min at 37 C. Cells were vigorously pipette to disrupt nonspecific conjugates and CD4 expression on B220+ cells was analyzed on a FACSCanto II (BD Biosciences).

Recombinant:

Article Title: OTUD1 deubiquitylase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways
Article Snippet: .. The following reagents were obtained as indicated: human TNF-α and IL-1β (BioLegend), poly(dA:dT) and high molecular weight poly(I:C) (Invivogen), LPS from E. coli O111:B4 (Invivogen), cycloheximide (Sigma-Aldrich), recombinant mouse CD40 ligand (R&D Systems), α -IgM (Southern Biotech), zVAD-FMK (ZVAD, Peptide Institute, Inc.), and necrostatin-1 (ENZO Life Sciences). ..

Article Title: The mTOR-Bach2 Cascade Controls Cell Cycle and Class Switch Recombination during B Cell Differentiation
Article Snippet: .. B1-8 hi splenic B cells were stimulated with IL-2 (100 U/ml), recombinant mouse IL-4 (5 ng/ml), recombinant mouse IL-5 (1.5 ng/ml), recombinant mouse CD40 ligand (0.2 ng/ml) (all R&D Systems), and NP(40)-Ficoll (0.01 ng/ml; Biosearch Technologies Inc.), and 0.5 nM rapamycin or 10 nM AZD8055 was supplemented in culture medium. ..

Article Title: OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways
Article Snippet: .. The following reagents were obtained as indicated: human TNF-α and IL-1β (BioLegend), poly(dA:dT) and high molecular weight poly(I:C) (Invivogen), LPS from E. coli O111:B4 (Invivogen), cycloheximide (Sigma-Aldrich), recombinant mouse CD40 ligand (R&D Systems), a-IgM (Southern Biotech), zVAD-FMK (ZVAD, Peptide Institute, Inc.), N-acetylcysteine (NAC, Wako-Fujifilm), Mito-TEMP (MedChemExpress), butylated hydroxyanisole (BHA, Santa Cruz), and necrostatin-1 (ENZO Life Sciences). ..

Flow Cytometry:

In Vitro:

Incubation:

High Molecular Weight:

Image Search Results

Heme promotes plasma cell (PC) formation in response to T-dependent and T-independent activating signals. (A) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and the indicated concentration of heme or vehicle was added after 24 h. Representative flow cytometry analysis at 72 h. (B) Quantitation of the frequency of CD138 + PCs from (A). (C) Naïve B cells were stimulated with CD40L, IL-4, and IL-5 signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry analysis at 48, 72, and 96 h. (D) Quantitation of the frequency of CD138 + PCs from (C). (E) Naïve B cells were stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry analysis at the indicated time points. (F) Quantitation of the frequency of CD138 + PCs from (E). Data represent the combination of at least 2 independent experiments with at least 3 mice each. Error bars represent the mean ± SD and each data point an individual mouse. Statistics were calculated using a paired Student t- test.

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme promotes plasma cell (PC) formation in response to T-dependent and T-independent activating signals. (A) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and the indicated concentration of heme or vehicle was added after 24 h. Representative flow cytometry analysis at 72 h. (B) Quantitation of the frequency of CD138 + PCs from (A). (C) Naïve B cells were stimulated with CD40L, IL-4, and IL-5 signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry analysis at 48, 72, and 96 h. (D) Quantitation of the frequency of CD138 + PCs from (C). (E) Naïve B cells were stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry analysis at the indicated time points. (F) Quantitation of the frequency of CD138 + PCs from (E). Data represent the combination of at least 2 independent experiments with at least 3 mice each. Error bars represent the mean ± SD and each data point an individual mouse. Statistics were calculated using a paired Student t- test.

Article Snippet: B cells were stimulated with either 20 μg/mL LPS (L2630; Sigma), 20 ng/mL IL-2 (575406; BioLegend), and 5 ng/mL IL-5 (581504; BioLegend) or 500 ng/mL CD40L (R&D Systems, 8230-CL), 10 ng/mL IL-4 (R&D Systems, 404-ML), and 10 ng/mL IL-5.

Techniques: Clinical Proteomics, Ex Vivo, Concentration Assay, Flow Cytometry, Quantitation Assay, Control

Heme induces more proliferating cells at later divisions. (A) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and the indicated concentration of heme or vehicle was added after 24 h. Representative flow cytometry histograms showing CTY dilution at 72 h for all B cells in culture. (B) Quantitation of the mean division number (MDN) for all cultures in (A). (C) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, IL-5) signals ex vivo and 60 µM of heme or vehicle control added after 24 h. Representative flow cytometry histograms showing CTY dilution at the indicated time points for all B cells in culture. (D) Quantitation of the MDN of cell cultures in (C). (E) Representative flow cytometry analysis of CD138 + cells vs CTY dilution at the indicated time points. (F) Quantitation of the percentage of CD138 + cells within each division from (E). (G) Naïve B cells were stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry histograms showing CTY dilution at 72 h. (H) Quantitation of the percentage of CD138 + cells within each division from (G). All data represent the combination of at least 2 independent experiments. Data points in (B) and (D) represent individual mice with horizontal bars depicting the data mean. Error bars in (F) and (H) depict mean ± SD. Statistics were calculated using a paired Student t- test.

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme induces more proliferating cells at later divisions. (A) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and the indicated concentration of heme or vehicle was added after 24 h. Representative flow cytometry histograms showing CTY dilution at 72 h for all B cells in culture. (B) Quantitation of the mean division number (MDN) for all cultures in (A). (C) Naïve B cells were stimulated with T-dependent (CD40L, IL-4, IL-5) signals ex vivo and 60 µM of heme or vehicle control added after 24 h. Representative flow cytometry histograms showing CTY dilution at the indicated time points for all B cells in culture. (D) Quantitation of the MDN of cell cultures in (C). (E) Representative flow cytometry analysis of CD138 + cells vs CTY dilution at the indicated time points. (F) Quantitation of the percentage of CD138 + cells within each division from (E). (G) Naïve B cells were stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo, and 60 µM of heme or vehicle control was added after 24 h. Representative flow cytometry histograms showing CTY dilution at 72 h. (H) Quantitation of the percentage of CD138 + cells within each division from (G). All data represent the combination of at least 2 independent experiments. Data points in (B) and (D) represent individual mice with horizontal bars depicting the data mean. Error bars in (F) and (H) depict mean ± SD. Statistics were calculated using a paired Student t- test.

Techniques: Ex Vivo, Concentration Assay, Flow Cytometry, Quantitation Assay, Control

Heme augments S phase cells at later divisions. (A) Naïve B cells were stained with CTY and stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and 60 µM heme or vehicle control added after 24 h. BrdU was pulsed into the cultures 1 h prior to staining and analysis at 72 h. Representative flow cytometry histograms (left) and quantitation (right) showing CTY dilution vs BrdU incorporation. (B) Naïve B cells were stained with CTY and stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo and 60 µM heme or vehicle control added after 24h. BrdU was pulsed into the cultures 1 h prior to staining and analysis at 72 h. Representative flow cytometry histograms (left) and quantitation (right) showing CTY dilution vs BrdU incorporation. (C) Representative histogram (left) or MDN quantitation (right) of BrdU + cells from (A). (D) Representative histogram (left) or MDN quantitation (right) of BrdU + cells from (B). Data represent the combination of at least 2 independent experiments. Error bars represent the mean ± SD and each data point an individual mouse. Statistics were calculated using a paired Student t- test.

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme augments S phase cells at later divisions. (A) Naïve B cells were stained with CTY and stimulated with T-dependent (CD40L, IL-4, and IL-5) signals ex vivo and 60 µM heme or vehicle control added after 24 h. BrdU was pulsed into the cultures 1 h prior to staining and analysis at 72 h. Representative flow cytometry histograms (left) and quantitation (right) showing CTY dilution vs BrdU incorporation. (B) Naïve B cells were stained with CTY and stimulated with T-independent (LPS, IL-2, and IL-5) signals ex vivo and 60 µM heme or vehicle control added after 24h. BrdU was pulsed into the cultures 1 h prior to staining and analysis at 72 h. Representative flow cytometry histograms (left) and quantitation (right) showing CTY dilution vs BrdU incorporation. (C) Representative histogram (left) or MDN quantitation (right) of BrdU + cells from (A). (D) Representative histogram (left) or MDN quantitation (right) of BrdU + cells from (B). Data represent the combination of at least 2 independent experiments. Error bars represent the mean ± SD and each data point an individual mouse. Statistics were calculated using a paired Student t- test.

Techniques: Staining, Ex Vivo, Control, Flow Cytometry, Quantitation Assay, BrdU Incorporation Assay

Heme represses the p21-Rb cell cycle regulatory axis. (A) Model depicting how p53, p21, and Rb function to regulate the G1 to S phase transition of the cell cycle under conditions of cell cycle arrest (left) or cell cycle progression (right). Naïve B cells were stimulated with CD40L, IL-4, and IL-5, and heme or vehicle control was added at 24 h. (B) Bar plot representing Trp53 transcript abundance at 54 h and 72 h measured by RT-qPCR. (C) Western blot for p53 protein and β-actin. Images for p53 protein were adjusted for brightness (+40%) and contrast (−40%). (D) Bar plot representing p53 expression relative to β-actin from (C). (E) Boxplot quantitating the ATAC-seq signal at p53 motifs in the indicated condition. (F) Genome plot of the Cdkn1a locus depicting the ATAC-seq signal and significant differential accessible region (box) in heme- vs vehicle-treated activated B cells (ActB) and plasma cells (PCs). The location of a previously identified p53 binding site is indicated in red. (G) Bar plot representing Cdkn1a transcript abundance at 54 h and 72 h measured by RT-qPCR. (H) Representative flow cytometry histogram showing intracellular staining for p21 protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (I) Bar plot quantitating MFI of p21 expression from (H). (J) Representative flow cytometry histogram showing intracellular staining for Rb protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (K) Bar plot quantitating MFI of Rb expression from (J). Data represent the combination of at least 2 independent experiments with the indicated P value calculated with paired Student t- tests. FMO, Fluorescence Minus One; MFI, median fluorescence intensity; RPPM, reads per peak per million.

Journal: The Journal of Immunology Author Choice

Article Title: Heme enhances B-cell proliferation and plasma cell formation through reduced p21 and Rb expression

doi: 10.1093/jimmun/vkag025

Figure Lengend Snippet: Heme represses the p21-Rb cell cycle regulatory axis. (A) Model depicting how p53, p21, and Rb function to regulate the G1 to S phase transition of the cell cycle under conditions of cell cycle arrest (left) or cell cycle progression (right). Naïve B cells were stimulated with CD40L, IL-4, and IL-5, and heme or vehicle control was added at 24 h. (B) Bar plot representing Trp53 transcript abundance at 54 h and 72 h measured by RT-qPCR. (C) Western blot for p53 protein and β-actin. Images for p53 protein were adjusted for brightness (+40%) and contrast (−40%). (D) Bar plot representing p53 expression relative to β-actin from (C). (E) Boxplot quantitating the ATAC-seq signal at p53 motifs in the indicated condition. (F) Genome plot of the Cdkn1a locus depicting the ATAC-seq signal and significant differential accessible region (box) in heme- vs vehicle-treated activated B cells (ActB) and plasma cells (PCs). The location of a previously identified p53 binding site is indicated in red. (G) Bar plot representing Cdkn1a transcript abundance at 54 h and 72 h measured by RT-qPCR. (H) Representative flow cytometry histogram showing intracellular staining for p21 protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (I) Bar plot quantitating MFI of p21 expression from (H). (J) Representative flow cytometry histogram showing intracellular staining for Rb protein in heme or vehicle control cultures after 72 h along with isotype staining and FMO controls. (K) Bar plot quantitating MFI of Rb expression from (J). Data represent the combination of at least 2 independent experiments with the indicated P value calculated with paired Student t- tests. FMO, Fluorescence Minus One; MFI, median fluorescence intensity; RPPM, reads per peak per million.

Techniques: Sublimation, Control, Quantitative RT-PCR, Western Blot, Expressing, Clinical Proteomics, Binding Assay, Flow Cytometry, Staining, Fluorescence

Epitope-specific priming of CD8+ T cells by B cells and DCs resulted in different activation marker expression profiles. B cells and DCs isolated from the C57Bl/6 spleen were either LPS treated or not and then loaded with SIINFEKL peptide. After 48 h of incubation with CD8+ T cells in the presence or absence of GM-CSF and IL-4, flow cytometry was performed to measure the expression of activation markers ( A ) 4-1BB, ( B ) CD25, ( C ) CD27, ( D ) CD28, ( E ) CD40L, ( F ) FASR, ( G ) CD69, ( H ) Ki67, ( I ) OX40, and ( J ) ICOS. Median fluorescence intensities (MFI) were plotted with mean and standard deviation. The percentage of CD8+ T cells positive for the expression of the respective cell surface markers are shown in . Each treatment group was tested with five biological replicates and each data point on the plot represents a biological replicate. Asterisks * indicate p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Results are representative of at least two similar, independent experiments.

Journal: Vaccines

Article Title: CD8+ T Cells Primed by Antigenic Peptide-Pulsed B Cells or Dendritic Cells Generate Similar Anti-Tumor Response

doi: 10.3390/vaccines13090953

Figure Lengend Snippet: Epitope-specific priming of CD8+ T cells by B cells and DCs resulted in different activation marker expression profiles. B cells and DCs isolated from the C57Bl/6 spleen were either LPS treated or not and then loaded with SIINFEKL peptide. After 48 h of incubation with CD8+ T cells in the presence or absence of GM-CSF and IL-4, flow cytometry was performed to measure the expression of activation markers ( A ) 4-1BB, ( B ) CD25, ( C ) CD27, ( D ) CD28, ( E ) CD40L, ( F ) FASR, ( G ) CD69, ( H ) Ki67, ( I ) OX40, and ( J ) ICOS. Median fluorescence intensities (MFI) were plotted with mean and standard deviation. The percentage of CD8+ T cells positive for the expression of the respective cell surface markers are shown in . Each treatment group was tested with five biological replicates and each data point on the plot represents a biological replicate. Asterisks * indicate p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Results are representative of at least two similar, independent experiments.

Article Snippet: Recombinant mouse CD40L (Cat.# 8230-CL-050/CF) was purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Marker, Expressing, Isolation, Incubation, Flow Cytometry, Fluorescence, Standard Deviation

Characterization of early B‐cell activation using feeder‐free CD40L presentation. (a) Optical microscopy images (63×) of human naïve B‐cells on day‐0 and activated up to day‐4 in the presence of microbeads presenting CD40L (MB‐CD40L), IL‐4, and BAFF. (b) Plot comparing the single cell surface area of naïve B‐cells with the B‐cells after 18 h of activation via indicated methods of CD40L presentation. Each data point in the violin plot represents one single B‐cell. (c) Plot tracing the change in the single cell surface area of naïve B‐cells with that of the B‐cells activated by MB‐CD40L up to day‐4. (d) Plot comparing the percentage of single cells associated with MB‐CD40L on day‐0 (1 h) up to day‐3. (e) Plot comparing the percentage of single cells (with and without microbeads) on day‐0 with that after activation by MB‐CD40L up to day‐4. (f) Plot tracing the change in the surface area of B‐cell clusters formed by activating naïve B‐cells with MB‐CD40L from day‐1 up to day‐4. Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (*** p ≤ 0.001; **** p ≤ 0.0001; ns p > 0.05).

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Characterization of early B‐cell activation using feeder‐free CD40L presentation. (a) Optical microscopy images (63×) of human naïve B‐cells on day‐0 and activated up to day‐4 in the presence of microbeads presenting CD40L (MB‐CD40L), IL‐4, and BAFF. (b) Plot comparing the single cell surface area of naïve B‐cells with the B‐cells after 18 h of activation via indicated methods of CD40L presentation. Each data point in the violin plot represents one single B‐cell. (c) Plot tracing the change in the single cell surface area of naïve B‐cells with that of the B‐cells activated by MB‐CD40L up to day‐4. (d) Plot comparing the percentage of single cells associated with MB‐CD40L on day‐0 (1 h) up to day‐3. (e) Plot comparing the percentage of single cells (with and without microbeads) on day‐0 with that after activation by MB‐CD40L up to day‐4. (f) Plot tracing the change in the surface area of B‐cell clusters formed by activating naïve B‐cells with MB‐CD40L from day‐1 up to day‐4. Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (*** p ≤ 0.001; **** p ≤ 0.0001; ns p > 0.05).

Article Snippet: In this study, we used a recombinant CD40 ligand (Gly 116—Leu 261, CDL‐H82Db‐25ug; Acrobiosystems, USA) in an active trimer form that is enzymatically biotinylated on its N‐terminal Avi tag, which we will designate as soluble “monomer,” for this trimeric form is a single functional molecular unit.

Techniques: Activation Assay, Microscopy

Effects of interleukins on activation of human naïve B‐cells using MB‐CD40L. Plot comparing the yield of indicated phenotypes (a, b, d, and e) and fold expansions at the end of week‐1 and week‐2 (a and b) or week‐2 (c)–(e). (a) GC B‐cells (CD95 + CD38‐); (b) IgD‐ B‐cells; (c) fold expansion of live activated B‐cells compared to the number of seeded cells on day‐0; (d) memory B‐cells (IgD‐CD38‐/lo + CD27+); and (e) antibody‐secreting cells (ASCs) (IgD‐CD38++CD27+). Data were acquired from at least duplicates for each group each day and presented as mean + standard deviation. Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons for comparing week‐1 and week‐2 data and one‐way ANOVA with Tukey's multiple comparisons for comparing the end‐point results (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns p > 0.05). (f) Microscopy images acquired at 4× magnification of (i) human naïve B‐cells seeded into a 96‐well plate on day‐0 and activated by MB‐CD40L up to (ii) week‐1 and (iii) week‐2 in the presence of IL‐4, BAFF, IL‐2, IL‐10, and IL‐21 showing the growth of B‐cells and their clusters. (iv) 10×and (v) 63× magnification images. Darker contrasts originate from the microbeads, and lighter contrasts originate from the B‐cells.

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Effects of interleukins on activation of human naïve B‐cells using MB‐CD40L. Plot comparing the yield of indicated phenotypes (a, b, d, and e) and fold expansions at the end of week‐1 and week‐2 (a and b) or week‐2 (c)–(e). (a) GC B‐cells (CD95 + CD38‐); (b) IgD‐ B‐cells; (c) fold expansion of live activated B‐cells compared to the number of seeded cells on day‐0; (d) memory B‐cells (IgD‐CD38‐/lo + CD27+); and (e) antibody‐secreting cells (ASCs) (IgD‐CD38++CD27+). Data were acquired from at least duplicates for each group each day and presented as mean + standard deviation. Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons for comparing week‐1 and week‐2 data and one‐way ANOVA with Tukey's multiple comparisons for comparing the end‐point results (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns p > 0.05). (f) Microscopy images acquired at 4× magnification of (i) human naïve B‐cells seeded into a 96‐well plate on day‐0 and activated by MB‐CD40L up to (ii) week‐1 and (iii) week‐2 in the presence of IL‐4, BAFF, IL‐2, IL‐10, and IL‐21 showing the growth of B‐cells and their clusters. (iv) 10×and (v) 63× magnification images. Darker contrasts originate from the microbeads, and lighter contrasts originate from the B‐cells.

Techniques: Activation Assay, Standard Deviation, Microscopy

Functions of IL‐4 or IL‐21 signaling alone or together with IL‐2 and IL‐10 on the differentiation of naïve B‐cells into effector phenotypes. (a) Representative flow cytometry plots of CD27 versus CD38 for days 0, 4, 7, 10, and 14 of cultures of high‐purity naïve B‐cells activated by MB‐CD40L in the presence of indicated interleukin(s) or together with BAFF. Bar graphs for naïve B‐cell activation via MB‐CD40L with IL‐4 alone (medium orange), IL‐21 alone (light orange) or BAFF and ILs‐2, 4, 10, 21 (dark orange) comparing (b) fold expansion of B‐cells on day‐13 (c) the percentage of IgD‐ B‐cells from days 0 to 13 (d) the percentage of IgD‐IgG+ cells on day‐13, and the yield of (e) memory B‐cells and (f) antibody‐secreting cells (ASCs). (g) Median fluorescence intensity (MFI) of CD40 on activated B‐cells from days 0 to 13 for MB‐CD40L activation in the presence of IL‐4 only (medium orange), IL‐21 only (light orange) or BAFF and ILs‐2,4,10,21 (dark orange). Data were acquired as duplicates for two independent, healthy donors and presented as mean + standard deviation. Statistical analysis was conducted by applying ordinary one‐way ANOVA with Tukey's multiple comparisons (* p ≤ 0.05; ns p > 0.05).

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Functions of IL‐4 or IL‐21 signaling alone or together with IL‐2 and IL‐10 on the differentiation of naïve B‐cells into effector phenotypes. (a) Representative flow cytometry plots of CD27 versus CD38 for days 0, 4, 7, 10, and 14 of cultures of high‐purity naïve B‐cells activated by MB‐CD40L in the presence of indicated interleukin(s) or together with BAFF. Bar graphs for naïve B‐cell activation via MB‐CD40L with IL‐4 alone (medium orange), IL‐21 alone (light orange) or BAFF and ILs‐2, 4, 10, 21 (dark orange) comparing (b) fold expansion of B‐cells on day‐13 (c) the percentage of IgD‐ B‐cells from days 0 to 13 (d) the percentage of IgD‐IgG+ cells on day‐13, and the yield of (e) memory B‐cells and (f) antibody‐secreting cells (ASCs). (g) Median fluorescence intensity (MFI) of CD40 on activated B‐cells from days 0 to 13 for MB‐CD40L activation in the presence of IL‐4 only (medium orange), IL‐21 only (light orange) or BAFF and ILs‐2,4,10,21 (dark orange). Data were acquired as duplicates for two independent, healthy donors and presented as mean + standard deviation. Statistical analysis was conducted by applying ordinary one‐way ANOVA with Tukey's multiple comparisons (* p ≤ 0.05; ns p > 0.05).

Techniques: Flow Cytometry, Activation Assay, Fluorescence, Standard Deviation

Comparison of feeder‐free CD40 activation methods on expansion and differentiation of human naïve B‐cells. Bar graphs comparing the yield of (a) GC B‐cells, (b) fold expansion in number of total B‐cells, (c) memory B‐cell yield, and (d) ASC yield on days‐7 and 13 for naïve B‐cells (gray) activated with soluble monomeric CD40L (blue), anti‐human CD40 antibody (red), soluble multimeric CD40L (dark cyan) and MB‐CD40L (orange). Data were acquired each day as duplicates for each group and presented as mean + standard deviation. Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (* p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001; ns p > 0.05).

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Comparison of feeder‐free CD40 activation methods on expansion and differentiation of human naïve B‐cells. Bar graphs comparing the yield of (a) GC B‐cells, (b) fold expansion in number of total B‐cells, (c) memory B‐cell yield, and (d) ASC yield on days‐7 and 13 for naïve B‐cells (gray) activated with soluble monomeric CD40L (blue), anti‐human CD40 antibody (red), soluble multimeric CD40L (dark cyan) and MB‐CD40L (orange). Data were acquired each day as duplicates for each group and presented as mean + standard deviation. Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (* p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001; ns p > 0.05).

Techniques: Comparison, Activation Assay, Standard Deviation

Synergistic activation by MB‐CD40L, BCR, and TLR‐9 signaling for improved B‐cell expansion and differentiation. (a) Representative flow cytometry plot of CD27 versus CD38 for days 0–13 cells (gated on IgD‐ B‐cells from day‐4 onwards) for the activation of naïve B‐cells by MB‐CD40L with or without the B‐cell receptor signal by anti‐human IgM/G/A (H + L) antibody plus co‐receptor signal by anti‐human CD21 antibody (referred to as BCR) and/or toll‐like receptor signal by the TLR‐9 agonist CpG ODN 2006 in the presence of BAFF and the interleukins Il‐2, ‐4, ‐10 and ‐21. Bar graphs of (b) fold expansion and the yield of (c) memory B‐cells and (d) ASCs on day‐13 for each group. Data were acquired each day as duplicates for each group and represented as mean + standard deviation. Statistical analysis was conducted by applying ordinary one‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns p > 0.05).

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Synergistic activation by MB‐CD40L, BCR, and TLR‐9 signaling for improved B‐cell expansion and differentiation. (a) Representative flow cytometry plot of CD27 versus CD38 for days 0–13 cells (gated on IgD‐ B‐cells from day‐4 onwards) for the activation of naïve B‐cells by MB‐CD40L with or without the B‐cell receptor signal by anti‐human IgM/G/A (H + L) antibody plus co‐receptor signal by anti‐human CD21 antibody (referred to as BCR) and/or toll‐like receptor signal by the TLR‐9 agonist CpG ODN 2006 in the presence of BAFF and the interleukins Il‐2, ‐4, ‐10 and ‐21. Bar graphs of (b) fold expansion and the yield of (c) memory B‐cells and (d) ASCs on day‐13 for each group. Data were acquired each day as duplicates for each group and represented as mean + standard deviation. Statistical analysis was conducted by applying ordinary one‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns p > 0.05).

Techniques: Activation Assay, Flow Cytometry, Standard Deviation

Switching interleukins and CD40L dosage for the terminal differentiation of naïve B‐cells for effective generation of functional ASCs. (a) Representative flow cytometry plot of CD27 versus CD38 gated on IgD‐ B‐cells on day‐10 and day‐13. High‐purity naïve B‐cells were activated with MB‐CD40L, BAFF, anti‐BCR/CD21 antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for initial 10 days. On day‐10, CD40L dosage was either maintained (MB‐CD40L) or reduced by 10‐fold (MB‐CD40L low ). For each CD40L dosage, three different conditions were tested up to day‐13: All factors or all factors except IL‐21 or all factors except anti‐BCR antibodies and IL‐4. (b), (c) Bar graphs comparing the yield percentage of memory B‐cells (IgD‐CD38‐/lo + CD27) and ASCs (IgD‐CD38++CD27+) on day‐13 from indicated conditions. Data were acquired as duplicates and presented as mean + standard deviation. (d) Number of ASCs generated by day‐13 from the culture of 10,000 naïve B‐cells (day‐0). Either MB‐CD40L or Sol Multimer CD40L was employed for the entirety of the culture period. Other indicated culture conditions were varied between day‐10 and day‐13. Data were acquired as triplicates and presented as mean + standard deviation. (e), (f) The concentrations of antibodies of different isotypes, IgM and IgG, secreted by ASCs generated by activation of high‐purity naïve B‐cells using the indicated method of CD40L presentation in the presence of BAFF, anti‐BCR antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for the initial 10 days. On day‐10, cells were reseeded at 10,000 cells per well, CD40L dosage was reduced by 10‐fold, and all soluble factors except anti‐BCR antibodies and IL‐4 were employed. ELISA was performed using supernatant samples harvested on day‐12. Data were acquired as triplicates and presented as mean + standard deviation. (b)−(f) Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001; ns p > 0.05).

Journal: Bioengineering & Translational Medicine

Article Title: Microbead‐based synthetic niches for in vitro expansion and differentiation of human naïve B‐cells

doi: 10.1002/btm2.10751

Figure Lengend Snippet: Switching interleukins and CD40L dosage for the terminal differentiation of naïve B‐cells for effective generation of functional ASCs. (a) Representative flow cytometry plot of CD27 versus CD38 gated on IgD‐ B‐cells on day‐10 and day‐13. High‐purity naïve B‐cells were activated with MB‐CD40L, BAFF, anti‐BCR/CD21 antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for initial 10 days. On day‐10, CD40L dosage was either maintained (MB‐CD40L) or reduced by 10‐fold (MB‐CD40L low ). For each CD40L dosage, three different conditions were tested up to day‐13: All factors or all factors except IL‐21 or all factors except anti‐BCR antibodies and IL‐4. (b), (c) Bar graphs comparing the yield percentage of memory B‐cells (IgD‐CD38‐/lo + CD27) and ASCs (IgD‐CD38++CD27+) on day‐13 from indicated conditions. Data were acquired as duplicates and presented as mean + standard deviation. (d) Number of ASCs generated by day‐13 from the culture of 10,000 naïve B‐cells (day‐0). Either MB‐CD40L or Sol Multimer CD40L was employed for the entirety of the culture period. Other indicated culture conditions were varied between day‐10 and day‐13. Data were acquired as triplicates and presented as mean + standard deviation. (e), (f) The concentrations of antibodies of different isotypes, IgM and IgG, secreted by ASCs generated by activation of high‐purity naïve B‐cells using the indicated method of CD40L presentation in the presence of BAFF, anti‐BCR antibodies, TLR‐9 agonist, and IL‐2, IL‐4, IL‐10, and IL‐21 for the initial 10 days. On day‐10, cells were reseeded at 10,000 cells per well, CD40L dosage was reduced by 10‐fold, and all soluble factors except anti‐BCR antibodies and IL‐4 were employed. ELISA was performed using supernatant samples harvested on day‐12. Data were acquired as triplicates and presented as mean + standard deviation. (b)−(f) Statistical analysis was conducted by applying two‐way ANOVA with Tukey's multiple comparisons (* p < 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001; ns p > 0.05).

Techniques: Functional Assay, Flow Cytometry, Standard Deviation, Generated, Activation Assay, Enzyme-linked Immunosorbent Assay

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