Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet: Stringent ACE2 requirement for pancreatic islet cell infection with SARS-CoV-2 (A) Representative contour plots gated on live α, β, and “other” cells pre-treated with IgG (irrelevant polyclonal goat antibody AF7197) or the anti-ACE2 blocking antibody AF933 prior to SARS-CoV-2 infection (48 h). (B) Summary of SARS-CoV-2 NP expression by live islet cell subsets as a function of IgG treatment or ACE2 blockade (n = 6 donors). (C) Percent infection inhibition for β and “other” cells (inhibition for α cells is not shown because the very low extent of α cell infection in IgG-treated cultures for 2 of 6 donors substantially skews such calculations). (D) Infectious SARS-CoV-2 titers and extent of infection inhibition following ACE2 blockade (n = 3 donors). (E) Quantification of chemokines and cytokines in UV-inactivated TCS of SARS-CoV-2-infected islet cell cultures under conditions of IgG treatment or ACE2 blockade (48-h infection, n = 3 donors). (F) Infectious SARS-CoV-2 titers in TCS as a function of glucose concentration in islet culture medium (n = 3 donors). (G) Quantification of CXCL10 and CXCL11 in TCS as a function of glucose concentration. All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable.

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Infection, Blocking Assay, Expressing, Inhibition, Concentration Assay

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet:

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Conjugation Assay, Purification, Blocking Assay, Recombinant, Control, Virus, Saline, Modification, Staining, Library Quantification, Antibody Labeling, Flow Cytometry, Software, Cytometry, Sequencing

Journal: Cell Death & Disease

Article Title: Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

doi: 10.1038/cddis.2017.351

Figure Lengend Snippet: Immunoglobulin isotypes in serum from SAMR1 and SAMP8 mice determined by ELISA. ( a ) Serum IgM and IgG1 from 2- and 10-month-old SAMR1 and SAMP8 mice. ( b ) IgG2a, IgG2b and IgG3 isotypes were quantified on sera from 10-month-old SAMP8 and SAMR1 mice. Data are measurements of individual mice. Means are indicated by the horizontal lines. Comparisons were made with the unpaired two-tailed Student’s t -test: ** P <0.01; *** P <0.001

Article Snippet: Standard curves for each Ig isotype were generated using purified myeloma proteins or antibodies: IgM (clone G155-228, BD Biosciences), IgG1 (clone 4.19, in-house), IgG2a (clone PK136, in-house), IgG2b (clone Y.3, in-house), IgG3 (clone MG3-3S, Biolegend, San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

doi: 10.1038/cddis.2017.351

Figure Lengend Snippet: Plasma cell B cell maturation and distribution of the memory B-cell compartments in aged SAMP8 mice. ( a ) Cells were stained with anti-CD19-Violet-421, anti-CD45R-PE and anti-CD138-APC. Absolute numbers of CD138 + plasmablasts were calculated from flow cytometry as in . Data are mean±S.E.M., ( n =4). ( b ) IgG1-specific ELISPOT analysis (see ‘Materials and Methods’) of purified samples of B1REL and B2 cells obtained from aged SAMR1 and SAMP8 mice. The numbers of IgG1- antibody secreting cells (ASCs) are shown. Data are mean±S.E.M., ( n =4). ( c ) RT-qPCR analyses were performed for Pax5, Xbp-1, Blimp1 and AID transcript expression in purified B1REL cells from 10-month-old SAMR1 and SAMP8 mice (see ‘Materials and Methods’). The Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA) was used to calculate the C T of each reaction. The amount of specific transcripts in each cDNA sample was determined as the 2 −ΔΔ C T , relative to that of the HPRT transcripts and normalized to those obtained for 2-month-old BALB/c samples used as reference. Data are mean±S.E.M. ( n =6–10 performed in duplicates). ( d–f ) Flow cytometry analyses 10-month-old SAMR1 and SAMP8 mice spleen samples. Representative dot-plots are shown for each mouse. Numbers inside the plots are frequencies of each population (mean±S.E.M.: n =4 for T FH cells and for GL7 + and IgD+ cells, and n =3 for memBC). Fluorescence scales are logarithmic. ( d ) Staining was performed with anti-CD4-APC, anti-CXCR5-Violet-421 and anti-PD1-biotynilated and revealed with streptavidin-PE-Cy7. T FH cells were gated as CD4 + CXCR5 + PD1 + cells (boxes). ( e ) MemBC were determined as CD11b − Gr1 − CD138 − IgM − IgD − CD19 + CD38 + IgG1 + by using anti-CD11b- and anti-Gr1-PE-Cy7, anti-CD138-PE, anti-IgM- and anti-IgD-FITC, anti-CD19-Violet-421, anti-CD38-APC and rat anti-mouse IgG1-biotynilated (clone A85-1, rat IgG1/k). The FMO control was a biotinylated rat IgG1/ k (clone R3-34). Biotynilated Abs were revealed with streptavidin-APC-Cy7. The histograms display the IgG1 + cells among the CD138 − CD38 + cells (empty, FMO isotype control; filled gray, IgG1 + cells). ( f ) Staining was performed with anti-CD19-Violet-421, anti-GL7-PE and anti-IgD-FITC, to determine GL7 + cells and naive IgD + cells on gated CD19 cells. ( g ) Bar graph shows the absolute number/spleen of T FH , GL7 + , memBC and naive B cells (right Y scale). These absolute numbers were calculated from the frequencies of each population. Shown are the mean±S.E.M. ( n =4 and 3 for memBC). The group comparisons were made using the unpaired two-tailed Student’s t -test: * P <0.05

Article Snippet: Standard curves for each Ig isotype were generated using purified myeloma proteins or antibodies: IgM (clone G155-228, BD Biosciences), IgG1 (clone 4.19, in-house), IgG2a (clone PK136, in-house), IgG2b (clone Y.3, in-house), IgG3 (clone MG3-3S, Biolegend, San Diego, CA, USA).

Techniques: Clinical Proteomics, Staining, Flow Cytometry, Enzyme-linked Immunospot, Purification, Quantitative RT-PCR, Expressing, Software, Fluorescence, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Altered marginal zone and innate-like B cells in aged senescence-accelerated SAMP8 mice with defective IgG1 responses

doi: 10.1038/cddis.2017.351

Figure Lengend Snippet: In vitro and in vivo responses of B-cell subsets from SAMP8 mice stimulated with LPS ( a ) and ( b ). The indicated B-cell subsets identified as in from spleens of 10-month-old SAMP8 and SAMR1 mice were FACS-purified, and then labeled with the CellTrace Violet kit before culturing for 72 h in the presence or absence of LPS (see Materials and Methods). After culture, the cells were washed and stained to detect CD138 and the incorporated violet dye by flow cytometry. ( a ) Representative results from B1REL, ABC and B2 cells after LPS stimulation are shown. Fluorescence scales are logarithmic. The numbers in the plots are the frequency in each quadrant. Data are means±S.E.M. ( n =3). ( b ) The IgM and IgG1 secreted into the culture medium after 72 h were determined by ELISA. Data are means±S.E.M. ( n =3). Comparisons were made using a two-tailed Student’s t -test: * P <0.05. ( c–e ) 10-month-old SAMR1 and SAMP8 mice were injected i.p. with LPS and after 3, 5 and 7 days serum and cell suspensions from spleens were obtained. ( c ) The IgM and IgG1 levels in sera were determined by ELISA. ( d ) Absolute numbers of B1REL and B2 cells (determined as in ) are shown. ( e ) Left, representative contour plots of splenic cells stained with anti-CD19-PE, anti-CD45R-FITC, anti-IgM-PE-Cy7 and anti-CD138-byotinilated revealed with streptavidin-APC at day 7 post-injection. B1REL cells were identified as in , and electronically gated to show the distribution of CD138 and IgM on them. The gates within the plots identify CD138 + IgM − and CD138 + IgM + cells. The numbers are the frequencies of these cells. Right, absolute numbers of CD138 + IgM − and CD138 + IgM + B1REL cells were calculated at the indicated times after LPS injection. The data were obtained from the frequencies of each cell population (means±S.E.M., n =3–4). Comparisons were made with the unpaired two-tailed Student’s t -test: * P <0.05; ** P <0.01. ( f ) Titration of anti-peptide-specific IgGs after in vivo immunizations with peptide-OVA. Aged SAMP8 ( n =5) and SAMR1 ( n =3) were immunized with peptide-OVA (see ‘Materials and Methods’), and the amounts of IgG specific for the peptide were determined by indirect ELISA in serial dilutions of the sera. Shown are the data for each individual mouse

Article Snippet: Standard curves for each Ig isotype were generated using purified myeloma proteins or antibodies: IgM (clone G155-228, BD Biosciences), IgG1 (clone 4.19, in-house), IgG2a (clone PK136, in-house), IgG2b (clone Y.3, in-house), IgG3 (clone MG3-3S, Biolegend, San Diego, CA, USA).

Techniques: In Vitro, In Vivo, Purification, Labeling, Staining, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Injection, Titration, Indirect ELISA