Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: CD38 expression and activity increase at late stages of ZIKV infection and correlate with NAD + decline in the brain (A) Total NADase activity measured in the brains of ZIKV-infected and mock-injected mice over the course of infection, showing significant increases from 18 dpi onward ( n ≥ 7 mice per group). (B) Overlay of NADase activity (red line) and NAD + levels (gray line), both relative to mock controls (dashed line), showing an inverse temporal association. (C–H) Relative mRNA expression of Sarm1 , Cd157 , and Cd38 , respectively, in the brains of ZIKV-infected and control mice ( n ≥ 4 mice per group). (D, F, H) Overlays of mRNA expression profiles of Sarm1 (D), Cd157 (F), and Cd38 (H) with NAD + levels (gray line) and ZIKV genomic RNA (yellow line), indicating that Cd38 induction temporally coincides with NAD + decline, while Sarm1 and Cd157 do not. (I) Linear regression shows a positive correlation between total NADase activity and Cd38 mRNA expression ( n = 50). (J) CD38-dependent NADase activity, calculated as the fraction inhibited by the specific CD38 inhibitor 78c ( n ≥ 6 mice per group). (K) CD38-independent NADase activity, which remains low and unchanged during infection ( n ≥ 6 mice per group). (L) On the right, representative Western blot of CD38 protein expression in brain extracts at 24 dpi, with α-tubulin as loading control (representative bands from the same experiment shown in the full blot in ). On the left, quantification of the Western blot bands’ intensities (CD38/α-tubulin) relative to mock ( n ≥ 4 mice per group). Data in panels A, C, E, G, J, K, and L are presented as mean ± SD; panels B, D, F, and H as mean ± SEM (shaded area). Statistical significance was determined by unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Expressing, Activity Assay, Infection, Injection, Control, Western Blot, MANN-WHITNEY

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: CD38 inhibition prevents NAD + depletion in the brains of ZIKV-infected mice (A) Schematic representation of the experimental design. Neonatal mice were subcutaneously infected with ZIKV at postnatal day 3 (P3). At 21 days post-infection (dpi), animals received a unilateral intracerebroventricular (i.c.v.) injection of the CD38-blocking antibody Ab68 (5.76 μg) or vehicle (Veh), and brains were collected at 24 dpi for analysis. (B) NAD + hydrolase activity in brain tissue. (C) Quantification of total NAD + levels in brain tissue. Data are presented as mean ± SD. Statistical analyses were performed using an unpaired Student’s t test ( n ≥ 7 mice per group). ∗p ≤ 0.05; ∗∗∗∗p ≤ 0.0001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Inhibition, Infection, Injection, Blocking Assay, Activity Assay

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: NAMPT is induced in the brain of ZIKV-infected mice (A) Relative mRNA expression of Nampt in the brains of ZIKV-infected and mock-injected mice across time points post-infection ( n ≥ 4 mice per group). (B) Overlay of Nampt mRNA expression (blue line), total NADase activity (red line), NAD + levels (gray line), and ZIKV genomic RNA (yellow line), all relative to mock controls (dashed line). (C–F) Correlation analyses between Nampt mRNA expression and (C) ZIKV genomic RNA, (D) Parp12 , (E) Parp10 , and (F) Cd38 mRNA expression. Nampt shows a strong correlation with viral load and early-induced Parps , but a weak correlation with Cd38 . (G) Representative Western blot of NAMPT protein expression in the brains of ZIKV-infected and mock-injected mice at 24 days post-infection (dpi; representative bands from the same experiment shown in the full blot in ).. HPRT was used as a loading control. The right panel shows quantification of NAMPT protein levels (NAMPT/HPRT ratio), normalized to mock controls ( n ≥ 4 mice per group). Data in panels A and G are presented as mean ± SD; panel B as mean ± SEM (shaded area). Correlations were assessed by linear regression analysis. Statistical analyses were performed using an unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Infection, Expressing, Injection, Activity Assay, Western Blot, Control, MANN-WHITNEY

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: Proinflammatory cytokine expression precedes CD38 induction in the brains of ZIKV-infected mice (A, C, E) Relative mRNA expression of Il6 and Tnf (linear scale), and Ccl5/Rantes (Log 10 -transformed) in the brains of ZIKV-infected and mock-injected mice across time points post-infection ( n ≥ 3 mice per group). All three inflammatory mediators were significantly upregulated during the early and mid-stages of infection. (B, D, F) Overlay of Il6 (B), Tnf (D), and Rantes – log 10 scale (F) mRNA expression profiles (purple line) with Cd38 mRNA (red line), NAD + levels (gray line), and ZIKV genomic RNA (yellow line), all relative to mock controls (dashed line). (G) Brain IL-6 protein levels determined by ELISA ( n ≥ 5 mice per group). The temporal pattern shows that cytokine and chemokine induction precede Cd38 expression, suggesting that neuroinflammation may contribute to the upregulation of CD38. Data in panels A, C, and E are presented as mean ± SD; panels B, D, and F as mean ± SEM (shaded area). Statistical analyses were performed using an unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Expressing, Infection, Transformation Assay, Injection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: iScience

Article Title: CD38 is a key mediator of NAD + depletion in the brain of ZIKV-infected mice

doi: 10.1016/j.isci.2025.114018

Figure Lengend Snippet: Infiltrating immune cells contribute to increased CD38 expression in the brains of ZIKV-infected mice (A) On the left, representative dot plots show the gating strategy used to identify CD45 lo CD11b + (putative resting microglia), CD45 hi CD11b + (infiltrating myeloid cells), and CD45 + CD11b − (lymphoid cells) populations. On the right, graphs showing the frequency of each population in ZIKV-infected and mock-injected mice ( n ≥ 5 mice per group). (B) Representative dot plots and quantification of CD11b + TMEM119 + , CD11b + TMEM119 + , and CD11b + TMEM119 - populations in the brains of infected and control mice ( n ≥ 6 mice per group), with the respective graphs showing the frequency of each population. (C–E) Representative histograms and quantification of CD38 expression (median fluorescence intensity, MFI) in CD11b + TMEM119 + (C) CD11b − TMEM119 + (D), and CD11b + TMEM119 - (E) populations. (F and G) Gating and quantification of CD3 + T cells (F) and corresponding CD38 expression (G). (H and I) Gating and quantification of CD19 + B cells (H) and corresponding CD38 expression (I). All graphs represent mean ± SD. Gating was based on negative controls; histogram quantification was performed using MFI, and curves were normalized to unit area. Statistical analyses were performed using an unpaired Student’s t test or Mann–Whitney test, as appropriate. ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Proteins were separated by electrophoresis on 15% SDS–polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, California, USA), and probed with primary antibodies against CD38 (AF4947, R&D Systems, Minnesota, USA) or NAMPT (AG-20A-0034-C050, AdipoGen Life Sciences, California, USA), using concentrations recommended by the manufacturers and previously validated in-house.

Techniques: Expressing, Infection, Injection, Control, Fluorescence, MANN-WHITNEY

Journal: Biochemical and biophysical research communications

Article Title: The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD + decline.

doi: 10.1016/j.bbrc.2019.03.199

Figure Lengend Snippet: (A-B) Relative mRNA levels of senescence-related genes in HUVECs treated with (A) X-ray irradiation (IR) (n=7–9) or (B) γ-IR (n=4–8). (C) Relative mRNA levels of CD38 in HUVECs treated with X-ray (n=7–9) or -IR (n=6–7). (D) Relative mRNA levels of senescence-related genes in irradiated MEFs (n=6). Relative mRNA levels (A-D) were determined by qRT-PCR. (E) CD38 relative enzymatic activity in multiple independent irradiation-induced senescent MEFs (passage 5–9) (n=2–3). Data are mean ± SE, *p < 0.05 vs control.

Article Snippet: Western immunoblotting was performed as previously described in Tarrago et al [ 9 ] using the following antibodies: mouse CD38 (R&D Systems AF4947) and β-Tubulin (Abcam Ab 15568).

Techniques: Irradiation, Quantitative RT-PCR, Activity Assay, Control

Journal: Biochemical and biophysical research communications

Article Title: The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD + decline.

doi: 10.1016/j.bbrc.2019.03.199

Figure Lengend Snippet: (A) mRNA expression of selected SASP factors (IL-6, IL-8, MCP-1) in senescent HUVECs, determined by qRT-PCR (n=7–9). (B) CD38 activity (n=3–5) and (C) a representative immunoblot showing CD38 levels in BMDMs treated with various factors found in the SASP. Data are mean ± SE, *p < 0.05 vs control.

Article Snippet: Western immunoblotting was performed as previously described in Tarrago et al [ 9 ] using the following antibodies: mouse CD38 (R&D Systems AF4947) and β-Tubulin (Abcam Ab 15568).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Western Blot, Control

Journal: Biochemical and biophysical research communications

Article Title: The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD + decline.

doi: 10.1016/j.bbrc.2019.03.199

Figure Lengend Snippet: (A) Scheme showing production of control conditioned media (CM) and SASP (senescent cell conditioned media) from HUVECs or PDPs for treatment of non-senescent cells. (B) Relative CD38 mRNA expression and activity in BMDMs treated with CM or SASP from x-ray- (n=7–9) or γ-IR-treated (n=3) HUVECs. (C and D) CD38 mRNA expression and activity in BMDMs treated with CM or SASP from PDP originating from (patient 1 in C) or (patient 2 in D) (n=2–3). (E) Relative CD38 expression (n=4–6) in HUVECs treated with CM or SASP from γ-IR MEFs. Data are mean ± SE, *p < 0.05 vs control.

Article Snippet: Western immunoblotting was performed as previously described in Tarrago et al [ 9 ] using the following antibodies: mouse CD38 (R&D Systems AF4947) and β-Tubulin (Abcam Ab 15568).

Techniques: Control, Expressing, Activity Assay

Journal: Biochemical and biophysical research communications

Article Title: The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD + decline.

doi: 10.1016/j.bbrc.2019.03.199

Figure Lengend Snippet: (A) Representative immunoblots demonstrating levels of CD38 in BMDMs treated with CM (control conditional media) or SASP (x-ray- or γ-IR-induced senescent cell conditioned media) from PDPs or HUVECs. (B) mRNA expression of other M1 markers, namely IL-6, IL-8, and Nos2 (inducible nitric oxide synthase), in BMDMs treated with CM or SASP from PDPs. (C) Model of SASP-induced CD38 expression in macrophages. Data are mean ± SE, *p < 0.05 vs control.

Article Snippet: Western immunoblotting was performed as previously described in Tarrago et al [ 9 ] using the following antibodies: mouse CD38 (R&D Systems AF4947) and β-Tubulin (Abcam Ab 15568).

Techniques: Western Blot, Control, Expressing

Journal: Biochemical and biophysical research communications

Article Title: The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD + decline.

doi: 10.1016/j.bbrc.2019.03.199

Figure Lengend Snippet: TaqMan Gene Expression Assays (Human)

Article Snippet: Western immunoblotting was performed as previously described in Tarrago et al [ 9 ] using the following antibodies: mouse CD38 (R&D Systems AF4947) and β-Tubulin (Abcam Ab 15568).

Techniques: Gene Expression

Journal: The Journal of Experimental Medicine

Article Title: Antigen Receptor Engagement Turns off the V(D)J Recombination Machinery in Human Tonsil B Cells

doi:

Figure Lengend Snippet: Ligation-mediated (LM)-PCR assay for detecting signal breaks in human GC B cells. ( a ) Scheme for LM-PCR strategy to detect RSS DNA breaks within the human Ig κ light chain locus. A primary V κ J κ 1 rearrangement is represented at the κ locus, and after V(D)J recombinase activation, downstream J κ segments may be chosen for a secondary rearrangement; J κ 5 is depicted. Blunt signal ends at the nonamer–heptamer sequences ( triangles ) are ligated to BW linkers. Linker-ligated DNA molecules are then amplified by PCR using two sets of specific primers (see Materials and Methods). ( b ) J κ signal breaks in human GC B cells. LM-PCR for J H 6 ( top ) and J κ 5 ( middle ) signal breaks in human mononuclear bone marrow ( BM ) cells, naive CD38 − IgD + FM tonsil B cells, and CD38 + IgD − GC B cells ( left ) and in centroblasts CD38 + CD77 + and centrocytes CD38 + CD77 − ( right ). PCR products were visualized with locus-specific probes. PCR from a region overlapping the germline J κ 5 segment was used as a control for DNA loading ( bottom ).

Article Snippet: Alternatively, tonsil mononuclear cells were stained with PE-labeled mouse anti–human CD38 ( Becton Dickinson ) and rat anti–human CD77 (Immunotech) that was visualized with FITC-labeled sheep anti–rat IgM (Serotec, Ltd., Kidlington, UK) and sorted into three fractions: ( a ) CD38 + CD77 + centroblasts; ( b ) CD38 + CD77 − centrocytes; and ( c ) a CD38 − CD77 − fraction that contained both naive FM B cells and memory B cells.

Techniques: Ligation, Activation Assay, Amplification, Control

Journal: The Journal of Experimental Medicine

Article Title: Antigen Receptor Engagement Turns off the V(D)J Recombination Machinery in Human Tonsil B Cells

doi:

Figure Lengend Snippet: RAG, TdT, λ-like, and V-preB gene expression in human tonsil B cell subsets. RNA from FACS ® -sorted CD38 − IgD + FM B lymphocytes, CD38 − CD77 − FM and memory B cells, CD38 + CD77 + GC centroblasts, CD38 + CD77 − GC centrocytes, CD38 + IgD − total GC B cells, and mononuclear bone marrow ( BM ) cells was analyzed by semiquantitative RT-PCR and visualized with labeled oligonucleotide probes. Igβ RT-PCR was used as a B cell–specific mRNA loading control. PCR assays were designed to distinguish between genomic DNA and mRNA using primers on different exons. PhosphorImager analysis showed that there is a fivefold difference in the amount of RAG1 mRNA amplified between centroblasts and centrocytes.

Article Snippet: Alternatively, tonsil mononuclear cells were stained with PE-labeled mouse anti–human CD38 ( Becton Dickinson ) and rat anti–human CD77 (Immunotech) that was visualized with FITC-labeled sheep anti–rat IgM (Serotec, Ltd., Kidlington, UK) and sorted into three fractions: ( a ) CD38 + CD77 + centroblasts; ( b ) CD38 + CD77 − centrocytes; and ( c ) a CD38 − CD77 − fraction that contained both naive FM B cells and memory B cells.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Labeling, Control, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Antigen Receptor Engagement Turns off the V(D)J Recombination Machinery in Human Tonsil B Cells

doi:

Figure Lengend Snippet: ΨL cell surface expression on GC B cells. ( a ) Flow cytometric analysis were performed on human tonsil B cells using anti-CD38, anti–V-preB, anti-Igκ, or anti-Igλ. Two different patient samples are shown. ( b ) Flow cytometric analysis of immunized wild-type and λ5-targeted (λ5 −/− ) mouse spleen cells using anti-B220, anti-GL7, anti-κ, and anti-λ, and either LM34 or SL156 anti-λ5 antibodies .

Article Snippet: Alternatively, tonsil mononuclear cells were stained with PE-labeled mouse anti–human CD38 ( Becton Dickinson ) and rat anti–human CD77 (Immunotech) that was visualized with FITC-labeled sheep anti–rat IgM (Serotec, Ltd., Kidlington, UK) and sorted into three fractions: ( a ) CD38 + CD77 + centroblasts; ( b ) CD38 + CD77 − centrocytes; and ( c ) a CD38 − CD77 − fraction that contained both naive FM B cells and memory B cells.

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Antigen Receptor Engagement Turns off the V(D)J Recombination Machinery in Human Tonsil B Cells

doi:

Figure Lengend Snippet: Regulated expression of RAG , TdT , λ-like , and V-preB genes. ( a ) Human tonsil CD38 − IgD + FM or CD38 + IgD − GC B cells were sorted ( d0 ) and cultured on CD40L-transfected fibroblasts with or without anti-κ+λ (α Ig) at 10 μg/ml, or IL-2, IL-4, or IL-10 for 3 d ( d3 ). Gene expression was analyzed by RT-PCR as described above. ( b ) CD38 + IgD − GC B cells were cultured with CD40L alone or with CD40L and an irrelevant IgG control ( Control Ig ), or with intact anti-κ ( Ig α–κ ) or anti-λ ( Ig α–λ ) or both ( Ig α–κ + α–λ ) or with Fab′2 anti-κ or anti-λ ( Fab′2 α–κ + α–λ ). Antibody concentrations are indicated in micrograms per milliliter. ( c ) GC cells reanalyzed by flow cytometry using anti-CD38 and anti–V-preB mAbs after 3 d of culture with CD40L and the indicated antibodies. GC B cells cultured with Ig α–κ + α–λ or Fab′2 α–κ + α–λ antibodies contained 1.5–2-fold more B cells than those with cultured with control antibody, and the percentage of dead cells was 40–50% in all of cultures as determined by trypan blue exclusion.

Article Snippet: Alternatively, tonsil mononuclear cells were stained with PE-labeled mouse anti–human CD38 ( Becton Dickinson ) and rat anti–human CD77 (Immunotech) that was visualized with FITC-labeled sheep anti–rat IgM (Serotec, Ltd., Kidlington, UK) and sorted into three fractions: ( a ) CD38 + CD77 + centroblasts; ( b ) CD38 + CD77 − centrocytes; and ( c ) a CD38 − CD77 − fraction that contained both naive FM B cells and memory B cells.

Techniques: Expressing, Cell Culture, Transfection, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Flow Cytometry

Journal:

Article Title: Dissecting the human peripheral B-cell compartment with phage display-derived antibodies

doi: 10.1046/j.1365-2567.1999.00847.x

Figure Lengend Snippet: Flow cytometric analysis of tonsil B cells. Staining of tonsil B cells with PE-labelled anti-CD38 and FITC-labelled anti-IgD antibodies resolves three major populations: IgD+ CD38− naive B cells, IgD− CD38+ germinal centre B cells and IgD− CD38− memory B cells. Note the small population of IgD− CD38bright plasma cell precursors.

Article Snippet: The following monoclonal and polyclonal antibodies were used for double and triple immunofluorescent analysis: FITC-conjugated and biotinylated F(ab′) 2 fragments of a monoclonal antihuman IgD antibody (Southern Biotechnology Associates, Birmingham, AL); CD38-PE, CD19-FITC, CD3-FITC, CD19-PerCP (all from Becton Dickinson, San Jose, CA); CD38-FITC (Immunotech, Marseille, France); Tricolor-conjugated CD38 and CD19 monoclonal antibodies (Monosan, Uden, the Netherlands); unconjugated sheep anti-M13 used for indirect immunofluorescent stainings of phage antibodies (Pharmacia, Uppsala, Sweden); both FITC- and PE-conjugated F(ab′) 2 fragments of donkey antisheep antibodies (Jackson Immunoresearch, Westgrove, PA).

Techniques: Staining, Clinical Proteomics

Journal:

Article Title: Dissecting the human peripheral B-cell compartment with phage display-derived antibodies

doi: 10.1046/j.1365-2567.1999.00847.x

Figure Lengend Snippet: The mPhAbs from category IV bind to a subpopulation of peripheral B cells. (a) Staining pattern of mPhAbs of category IV on adult bone marrow (ABM), blood, spleen and tonsil lymphocytes, sequentially stained with a monoclonal antibody against CD19. (b) A triple immunofluorescence staining on tonsil B cells with phage antibody IV-7, anti-CD38 and anti-IgD. The left panel depicts the IgD versus CD38 staining on total tonsil B cells. The right panel depicts the position of the phage-positive cells within the IgD versus CD38 staining.

Article Snippet: The following monoclonal and polyclonal antibodies were used for double and triple immunofluorescent analysis: FITC-conjugated and biotinylated F(ab′) 2 fragments of a monoclonal antihuman IgD antibody (Southern Biotechnology Associates, Birmingham, AL); CD38-PE, CD19-FITC, CD3-FITC, CD19-PerCP (all from Becton Dickinson, San Jose, CA); CD38-FITC (Immunotech, Marseille, France); Tricolor-conjugated CD38 and CD19 monoclonal antibodies (Monosan, Uden, the Netherlands); unconjugated sheep anti-M13 used for indirect immunofluorescent stainings of phage antibodies (Pharmacia, Uppsala, Sweden); both FITC- and PE-conjugated F(ab′) 2 fragments of donkey antisheep antibodies (Jackson Immunoresearch, Westgrove, PA).

Techniques: Staining, Immunofluorescence

Journal:

Article Title: Dissecting the human peripheral B-cell compartment with phage display-derived antibodies

doi: 10.1046/j.1365-2567.1999.00847.x

Figure Lengend Snippet: The mPhAbs from category V bind to a subpopulation of tonsil and spleen B cells. (a) Staining pattern of mPhAbs of category V on adult bone marrow (ABM), blood, spleen and tonsil lymphocytes, sequentially stained with a monoclonal antibody against CD19. (b) A triple immunofluorescence staining on tonsil B cells with mPhAbV-3, anti-CD38 and anti-IgD. The left panel depicts the IgD versus CD38 staining on total tonsil B cells. The right panel depicts the position of the phage-positive cells within the IgD versus CD38 staining.

Article Snippet: The following monoclonal and polyclonal antibodies were used for double and triple immunofluorescent analysis: FITC-conjugated and biotinylated F(ab′) 2 fragments of a monoclonal antihuman IgD antibody (Southern Biotechnology Associates, Birmingham, AL); CD38-PE, CD19-FITC, CD3-FITC, CD19-PerCP (all from Becton Dickinson, San Jose, CA); CD38-FITC (Immunotech, Marseille, France); Tricolor-conjugated CD38 and CD19 monoclonal antibodies (Monosan, Uden, the Netherlands); unconjugated sheep anti-M13 used for indirect immunofluorescent stainings of phage antibodies (Pharmacia, Uppsala, Sweden); both FITC- and PE-conjugated F(ab′) 2 fragments of donkey antisheep antibodies (Jackson Immunoresearch, Westgrove, PA).

Techniques: Staining, Immunofluorescence

Journal: The Journal of Experimental Medicine

Article Title: Antigen Receptor Engagement Turns off the V(D)J Recombination Machinery in Human Tonsil B Cells

doi:

Figure Lengend Snippet: Ligation-mediated (LM)-PCR assay for detecting signal breaks in human GC B cells. ( a ) Scheme for LM-PCR strategy to detect RSS DNA breaks within the human Ig κ light chain locus. A primary V κ J κ 1 rearrangement is represented at the κ locus, and after V(D)J recombinase activation, downstream J κ segments may be chosen for a secondary rearrangement; J κ 5 is depicted. Blunt signal ends at the nonamer–heptamer sequences ( triangles ) are ligated to BW linkers. Linker-ligated DNA molecules are then amplified by PCR using two sets of specific primers (see Materials and Methods). ( b ) J κ signal breaks in human GC B cells. LM-PCR for J H 6 ( top ) and J κ 5 ( middle ) signal breaks in human mononuclear bone marrow ( BM ) cells, naive CD38 − IgD + FM tonsil B cells, and CD38 + IgD − GC B cells ( left ) and in centroblasts CD38 + CD77 + and centrocytes CD38 + CD77 − ( right ). PCR products were visualized with locus-specific probes. PCR from a region overlapping the germline J κ 5 segment was used as a control for DNA loading ( bottom ).

Article Snippet: Alternatively, tonsil mononuclear cells were stained with PE-labeled mouse anti–human CD38 ( Becton Dickinson ) and rat anti–human CD77 (Immunotech) that was visualized with FITC-labeled sheep anti–rat IgM (Serotec, Ltd., Kidlington, UK) and sorted into three fractions: ( a ) CD38 + CD77 + centroblasts; ( b ) CD38 + CD77 − centrocytes; and ( c ) a CD38 − CD77 − fraction that contained both naive FM B cells and memory B cells.

Techniques: Ligation, Activation Assay, Amplification, Control

Journal: The Journal of Experimental Medicine

Article Title: Antigen Receptor Engagement Turns off the V(D)J Recombination Machinery in Human Tonsil B Cells

doi:

Figure Lengend Snippet: RAG, TdT, λ-like, and V-preB gene expression in human tonsil B cell subsets. RNA from FACS ® -sorted CD38 − IgD + FM B lymphocytes, CD38 − CD77 − FM and memory B cells, CD38 + CD77 + GC centroblasts, CD38 + CD77 − GC centrocytes, CD38 + IgD − total GC B cells, and mononuclear bone marrow ( BM ) cells was analyzed by semiquantitative RT-PCR and visualized with labeled oligonucleotide probes. Igβ RT-PCR was used as a B cell–specific mRNA loading control. PCR assays were designed to distinguish between genomic DNA and mRNA using primers on different exons. PhosphorImager analysis showed that there is a fivefold difference in the amount of RAG1 mRNA amplified between centroblasts and centrocytes.

Article Snippet: Alternatively, tonsil mononuclear cells were stained with PE-labeled mouse anti–human CD38 ( Becton Dickinson ) and rat anti–human CD77 (Immunotech) that was visualized with FITC-labeled sheep anti–rat IgM (Serotec, Ltd., Kidlington, UK) and sorted into three fractions: ( a ) CD38 + CD77 + centroblasts; ( b ) CD38 + CD77 − centrocytes; and ( c ) a CD38 − CD77 − fraction that contained both naive FM B cells and memory B cells.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Labeling, Control, Amplification