anti mouse igm elisa kit  (Bethyl)

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: Multiple extracellular vesicle populations produced by WEHI-231 cells contain immunoglobulin M (IgM) (A) WEHI-231 cells were imaged by bright field microscopy at the time of plating (day 0, left) and after 24 h (day 1, right) in complete media. Cell clusters, interspersed with single cells, are indicated with a turquoise arrowhead. (B) WEHI-231 cells were evaluated by flow cytometry for expression of IgM, IgD, CD19, CD5, CD23, and CD43 (blue) compared to fluorescence minus one (FMO) stained control cells (gray). Data are representative of two independent experiments. (C) IgM in 2000 × g supernatant (2kG supernatant) from untreated or LPS (10 μg/mL) treated WEHI-231 cells at day 2 (columns 1,2) and at day 3 (columns 3,4) was determined by anti-mouse IgM ELISA. Data are from one (day 2) and two (day 3) independent experiments. Data are represented as mean ± SEM. (D) Conditioned media from untreated WEHI-231 cells, or WEHI-231 cells treated with 1 or 10 μg/mL LPS, was centrifuged at 2000 × g and IgM expression in the resulting supernatant (8 μL/well) was determined by SDS/PAGE and immunoblot. Presented are immunoblots for IgM in samples prepared with unreduced (left, upper) or reduced (left, lower) sample buffer. Right image displays a longer membrane exposure of the unreduced samples, with low molecular weight IgM indicated by turquoise arrowhead. Purified mouse IgM (25 ng) was run as a positive control. (E) Lysate preparations of whole cells, and extracellular vesicle (EV) populations isolated by differential ultracentrifugation (2 kG, 11,000 × g (11 kG), 110,000 × g (110kG)), from untreated or LPS (10 μg/mL) treated WEHI-231 cells were evaluated for IgM by unreduced immunoblot. Total protein was 1 μg for all samples. Purified mouse IgM (3.1 ng) was run as a positive control in a non-contiguous lane and is presented at the far right in both images. (F) IgM in 11 kG EV (left), 110 kG EV (center), and 110kG supernatant (right) was determined by ELISA under buffer only or buffer +0.25% TX100 conditions, as indicated. Data in (D) and (E) are representative of at least two independent experiments. See also Figure S1 .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Produced, Microscopy, Flow Cytometry, Expressing, Fluorescence, Staining, Control, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Membrane, Molecular Weight, Purification, Positive Control, Isolation

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: Monomeric IgM is enriched in WEHI-231 EV and is detected at a greater level following detergent solubilization (A) Equivalent amounts of 11kG (upper row) and 110kG (lower row) EVs isolated by differential ultracentrifugation were titrated and adsorbed onto aldehyde sulfate microbeads, stained for surface expression of IgM (or isotype control), canonical EV marker proteins, and negative control protein (CD4), and analyzed by flow cytometry. Beads incubated with buffer alone were included as a control. Histogram overlays display the fluorescence for each antigen (CD4, CD9, CD63, CD81, or IgM). The graph indicates the percentage of beads positive for IgM compared to beads stained with isotype control antibody. Data are representative of at least three independent experiments. (B) WEHI 231 cells (16 × 10 cm plates; 0.1 × 10 6 cells/mL media) were cultured for two days, after which EV were isolated by DUC, resuspended in 1.1 mL PBS and serially titrated at 1:10, and subsequently analyzed by nanoparticle tracking analysis. Displayed are the starting and ending cell number (left) and cell viability (right). (C) Histogram plots for particle diameters of 11kG (left) and 110kG (right) samples are presented. (D) Displayed are the particle concentration (left), mode diameter (center), and percentile distribution by particle size (right) of 11kG and 110kG samples. N = 2 independent experiments. (E) Whole cell and isolated EV (2kG, 11kG, 110kG) lysates (2.3 μg protein/well) were evaluated for IgM and CD9 expression by SDS/PAGE and immunoblot. Total protein (left) and immunoblot detection of IgM and CD9 under reduced (right, top) or unreduced (right, bottom) conditions are presented. A longer (upper) and shorter (lower) exposure are presented for the unreduced immunoblot. Turquoise arrowheads (total protein gel) indicate visible discrepancies in expression of protein species across the four lysate preparations. Purified IgM (2.5 ng) and 110kG supernatant (10.5 μL) were run as control samples. N = 2 independent experiments. (F) The amount of IgM in 11kG and 110 kG EV populations under buffer only (no TX100) and buffer +0.25% TX100 conditions (top graph) and the total IgM in 11kG plus 110 kG EV populations combined as compared to 110kG supernatant (bottom graph), determined by anti-mouse IgM ELISA, are presented. Data are representative of three independent experiments. ANOVA and Tukey’s post-hoc analysis was applied to data presented in (A) and (F) prior to direct comparison. ∗ = p < 0.05; ∗∗ = p < 0.005; ∗∗∗ = p < 0.001. Data are represented as mean ± SEM. See also Figures S2 and .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Isolation, Staining, Expressing, Control, Marker, Negative Control, Flow Cytometry, Incubation, Fluorescence, Cell Culture, Concentration Assay, SDS Page, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Comparison

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: WEHI-231 11kG and 110 kG EV populations fractionated by sucrose gradient centrifugation reveal correlated expression of IgM and CD81 EV were first isolated by differential ultracentrifugation and then subjected to density gradient fractionation by overlay of sucrose/PBS layers with decreasing density. Fractions were collected (F1 least dense to F6 most dense) and washed with buffer. An equivalent proportion of each resuspended fraction was subsequently utilized for downstream analyses. (A) Density gradient fractions of EV were subjected to PAGE and immunoblotted for IgM and CD81, as indicated. Purified mouse IgM was run as a positive control. The measured density (g/mL) of each fraction are shown (right graph). N = 2 independent experiments. (B) Density gradient fractions of EV were tested by anti-mouse IgM ELISA under buffer +0.25% TX100 conditions for 11kG (left) and 110kG (right) EV populations. (C) Density gradient fractions were adsorbed onto aldehyde sulfate microbeads, stained for IgM and CD81, and analyzed by flow cytometry. Dot plots for IgM (top), CD81 (middle), and isotype control (bottom) of fraction 3 are shown, with gates indicating the positive bead populations. The graphs (bottom) show the percentage of positive beads for all 11 kG EV (left) and 110 kG EV (right) fractions. (D) Biparametric dot plots of buffer (left), fraction 3 11 kG EV (center), and fraction 3 110 kG EV (right) samples displaying the expression of isotype control/CD81 or IgM/CD81 are presented. Data presented in (A)–(D) are representative of at least two independent experiments. Data are represented as mean ± SEM.

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Gradient Centrifugation, Expressing, Isolation, Fractionation, Purification, Positive Control, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Control

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: Splenic B cells from WT and μsKO mice release 11kG and 110 kG EV that contain low molecular weight IgM WT mouse splenic B cells were isolated by immunocapture magnetic bead negative selection and cultured unstimulated for 24 h, after which conditioned media was harvested for EV isolation and analysis. (A) Cell surface expression of IgM, CD9, CD63, and CD81 was determined by flow cytometry at the time of plating (day 0) and at harvest (day 1). Histogram overlays of cells stained for antigen (blue) and corresponding FMO stained cells (gray) are presented. (B) EV (11kG and 110kG) in day 1 conditioned media were isolated by differential ultracentrifugation (DUC) and then fractionated by sucrose gradient centrifugation. Following buffer wash, each fraction was resuspended in an equivalent volume and then resolved by SDS/PAGE for immunoblot detection of IgM and CD9, as indicated. A sample of day 1 WT splenic B cell lysate (3.75 μg protein) was run as a positive control. Data are representative of at least two independent experiments. (C) WT and μsKO splenic B cells were isolated by immunocapture magnetic bead negative selection and cultured unstimulated for 24 h, after which conditioned media was collected and cell lysates were prepared. EV were isolated from conditioned media by DUC and resuspended in an equivalent volume of buffer. Equivalent protein (cell lysates; 2 μg) or volume (2kG supernatant, 11 kG EV, 110 kG EV, post-110kG supernatant) for WT and μsKO samples was resolved by SDS/PAGE under unreduced conditions, followed by immunoblot detection of IgM and CD81. (D) The amount of IgM, as determined by anti-mouse IgM ELISA under buffer and buffer +0.25% TX100 conditions, is shown for 2kG supernatant (top), 11 kG EV (lower left), and 110 kG EV (lower right). Data in (C) and (D) are representative of two independent experiments. Data are represented as mean ± SEM. See also Figure S4 .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Molecular Weight, Isolation, Selection, Cell Culture, Expressing, Flow Cytometry, Staining, Gradient Centrifugation, SDS Page, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: EV isolated from WT and μsKO mouse peritoneal cavity fluid and peritoneal B cell cultures contain low molecular weight IgM (A) Peritoneal cavity washout fluid was obtained from WT and μsKO mice and subjected to DUC to isolate 2kG, 11kG, and 110 kG EV populations, which were then resuspended in an equivalent volume of buffer, resolved by SDS/PAGE under unreduced conditions, and immunoblotted for IgM and CD81, along with 2kG and 110kG supernatant samples. Data are representative of two independent experiments. (B) IgM expression in the peritoneal cavity samples described in (A) was determined by anti-mouse IgM ELISA under buffer +0.25% TX100 conditions. Results are shown for the EV samples (left) and 110kG supernatant (right). (C) Peritoneal B-1a cells (CD5 + B220 lo CD43 + CD23 − CD19 + ), peritoneal B2 (B2P) cells (CD5 - B220 high CD43 − CD23 + CD19 + ), and splenic B2 (B2 S) cells (CD5 - B220 high CD43 − CD23 high CD21 lo/− CD19 + ) were sorted from WT and μsKO, as detailed in materials and methods. The sorted B-1a, B2P, and B2 S cells were cultured in media alone or media + LPS (25 μg/mL) for 24-h, after which conditioned media was harvested and the 11kG and 110 kG EV populations were subsequently isolated by differential ultracentrifugation. EV samples (11 kG EV, left and 110 kG EV, right) were resuspended in an equivalent volume of buffer, resolved by SDS/PAGE under non-reducing conditions, and immunoblotted for IgM. Data are representative of at least two independent experiments. See also Figures S5 and .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Isolation, Molecular Weight, SDS Page, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: IgM contained in B cell-derived EV can bind antigen CH12 cells were cultured in media alone for two days, after which the conditioned media was subjected to differential ultracentrifugation (DUC) to isolate EV. The EV samples (2kG, 11kG, and 110kG) were resuspended in an equivalent volume of buffer for subsequent analysis by immunoblot and ELISA. (A) An equivalent proportion of each sample was resolved by SDS/PAGE under unreduced conditions, and immunoblotted for IgM and CD81. CH12 cell lysate (lane 1; 7.5 μg) and 110kG supernatant (lane 5), were run as controls. (B) The amount of IgM in each EV sample under buffer +0.25% TX100 conditions was determined by anti-mouse IgM ELISA. For (A) and (B), data are representative of two independent experiments. (C) CH12 cells were incubated with PtC liposomes for 30 min on ice at 1:2000 stock and at sequential 1:2 dilutions thereof, after which FITC fluorescence was determined by flow cytometry and GMI determined. The extreme left data point is the GMI FITC for CH12 cells incubated in buffer only. The turquoise arrowhead indicates the reagent concentration (1:5000 stock) utilized for subsequent experiments. (D) WEHI 231 and CH12 cells were stained with IgM APC and PtC liposome FITC reagent for flow cytometry analysis. Dot plots indicate FMO PtC liposome (first row), FMO IgM APC (second row), and complete stain (third row) fluorescence profiles of single, viable WEHI-231 (left column) and CH12 (right column) cells. (E) EV samples (11kG and 110kG) were isolated by DUC from CH12 conditioned media, resuspended in an equivalent volume of buffer, sequentially titrated 1:2, incubated with an identical volume of aldehyde sulfate microbeads, and subsequently stained with a fluorescent panel of IgM APC, CD81 BV241, CD4 PE/Cy5 and PtC liposome FITC reagent for flow cytometry analysis. GMI for each fluorophore is shown for the most concentrated EV preparation (extreme right data point) and each titrated sample (progressing left). GMI fluorescence of beads incubated in buffer only is indicated by the extreme left data point. Data are representative of two independent experiments. (F) EV (11kG and 110kG) were isolated from WEHI-231 and CH12 conditioned media by differential ultracentrifugation and then titrated 1:2, adsorbed onto an equivalent volume of aldehyde sulfate microbeads, and then stained with the reagent panel described above for flow cytometry analysis (11 kG EV, left graphs; 110kG right graphs). GMI for each fluorophore is shown for the most concentrated EV preparation (extreme right data point) and each titrated sample (progressing left). GMI fluorescence of beads incubated in buffer only is indicated by the extreme left data point. (G) EV (11kG and 110kG) were isolated from CH12 conditioned media by DUC and subjected to sucrose gradient fractionation. Equivalent volumes of resultant fractions were subjected to SDS/PAGE under non-reducing conditions and immunoblotted for IgM and CD81. Data are representative of two independent experiments. (H) Fraction 4 from 11 kG EV and fraction 3 from 110 kG EV were analyzed by flow cytometry following adsorption to aldehyde sulfate microbeads and staining with the reagent panel described above. GMI fluorescence for each fluorophore is shown. GMI fluorescence of beads incubated in buffer only is indicated by the extreme left data point. Data are represented as mean ± SEM.

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Derivative Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Incubation, Liposomes, Fluorescence, Flow Cytometry, Concentration Assay, Staining, Isolation, Fractionation, Adsorption

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet:

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Control, Recombinant, Liposomes, Red Blood Cell Lysis, Staining, Lysis, Protease Inhibitor, Purification, Cell Isolation, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Membrane, Microscopy, Flow Cytometry, Knock-Out, Software

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: Multiple extracellular vesicle populations produced by WEHI-231 cells contain immunoglobulin M (IgM) (A) WEHI-231 cells were imaged by bright field microscopy at the time of plating (day 0, left) and after 24 h (day 1, right) in complete media. Cell clusters, interspersed with single cells, are indicated with a turquoise arrowhead. (B) WEHI-231 cells were evaluated by flow cytometry for expression of IgM, IgD, CD19, CD5, CD23, and CD43 (blue) compared to fluorescence minus one (FMO) stained control cells (gray). Data are representative of two independent experiments. (C) IgM in 2000 × g supernatant (2kG supernatant) from untreated or LPS (10 μg/mL) treated WEHI-231 cells at day 2 (columns 1,2) and at day 3 (columns 3,4) was determined by anti-mouse IgM ELISA. Data are from one (day 2) and two (day 3) independent experiments. Data are represented as mean ± SEM. (D) Conditioned media from untreated WEHI-231 cells, or WEHI-231 cells treated with 1 or 10 μg/mL LPS, was centrifuged at 2000 × g and IgM expression in the resulting supernatant (8 μL/well) was determined by SDS/PAGE and immunoblot. Presented are immunoblots for IgM in samples prepared with unreduced (left, upper) or reduced (left, lower) sample buffer. Right image displays a longer membrane exposure of the unreduced samples, with low molecular weight IgM indicated by turquoise arrowhead. Purified mouse IgM (25 ng) was run as a positive control. (E) Lysate preparations of whole cells, and extracellular vesicle (EV) populations isolated by differential ultracentrifugation (2 kG, 11,000 × g (11 kG), 110,000 × g (110kG)), from untreated or LPS (10 μg/mL) treated WEHI-231 cells were evaluated for IgM by unreduced immunoblot. Total protein was 1 μg for all samples. Purified mouse IgM (3.1 ng) was run as a positive control in a non-contiguous lane and is presented at the far right in both images. (F) IgM in 11 kG EV (left), 110 kG EV (center), and 110kG supernatant (right) was determined by ELISA under buffer only or buffer +0.25% TX100 conditions, as indicated. Data in (D) and (E) are representative of at least two independent experiments. See also Figure S1 .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Produced, Microscopy, Flow Cytometry, Expressing, Fluorescence, Staining, Control, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot, Membrane, Molecular Weight, Purification, Positive Control, Isolation

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: Monomeric IgM is enriched in WEHI-231 EV and is detected at a greater level following detergent solubilization (A) Equivalent amounts of 11kG (upper row) and 110kG (lower row) EVs isolated by differential ultracentrifugation were titrated and adsorbed onto aldehyde sulfate microbeads, stained for surface expression of IgM (or isotype control), canonical EV marker proteins, and negative control protein (CD4), and analyzed by flow cytometry. Beads incubated with buffer alone were included as a control. Histogram overlays display the fluorescence for each antigen (CD4, CD9, CD63, CD81, or IgM). The graph indicates the percentage of beads positive for IgM compared to beads stained with isotype control antibody. Data are representative of at least three independent experiments. (B) WEHI 231 cells (16 × 10 cm plates; 0.1 × 10 6 cells/mL media) were cultured for two days, after which EV were isolated by DUC, resuspended in 1.1 mL PBS and serially titrated at 1:10, and subsequently analyzed by nanoparticle tracking analysis. Displayed are the starting and ending cell number (left) and cell viability (right). (C) Histogram plots for particle diameters of 11kG (left) and 110kG (right) samples are presented. (D) Displayed are the particle concentration (left), mode diameter (center), and percentile distribution by particle size (right) of 11kG and 110kG samples. N = 2 independent experiments. (E) Whole cell and isolated EV (2kG, 11kG, 110kG) lysates (2.3 μg protein/well) were evaluated for IgM and CD9 expression by SDS/PAGE and immunoblot. Total protein (left) and immunoblot detection of IgM and CD9 under reduced (right, top) or unreduced (right, bottom) conditions are presented. A longer (upper) and shorter (lower) exposure are presented for the unreduced immunoblot. Turquoise arrowheads (total protein gel) indicate visible discrepancies in expression of protein species across the four lysate preparations. Purified IgM (2.5 ng) and 110kG supernatant (10.5 μL) were run as control samples. N = 2 independent experiments. (F) The amount of IgM in 11kG and 110 kG EV populations under buffer only (no TX100) and buffer +0.25% TX100 conditions (top graph) and the total IgM in 11kG plus 110 kG EV populations combined as compared to 110kG supernatant (bottom graph), determined by anti-mouse IgM ELISA, are presented. Data are representative of three independent experiments. ANOVA and Tukey’s post-hoc analysis was applied to data presented in (A) and (F) prior to direct comparison. ∗ = p < 0.05; ∗∗ = p < 0.005; ∗∗∗ = p < 0.001. Data are represented as mean ± SEM. See also Figures S2 and .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Isolation, Staining, Expressing, Control, Marker, Negative Control, Flow Cytometry, Incubation, Fluorescence, Cell Culture, Concentration Assay, SDS Page, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Comparison

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: WEHI-231 11kG and 110 kG EV populations fractionated by sucrose gradient centrifugation reveal correlated expression of IgM and CD81 EV were first isolated by differential ultracentrifugation and then subjected to density gradient fractionation by overlay of sucrose/PBS layers with decreasing density. Fractions were collected (F1 least dense to F6 most dense) and washed with buffer. An equivalent proportion of each resuspended fraction was subsequently utilized for downstream analyses. (A) Density gradient fractions of EV were subjected to PAGE and immunoblotted for IgM and CD81, as indicated. Purified mouse IgM was run as a positive control. The measured density (g/mL) of each fraction are shown (right graph). N = 2 independent experiments. (B) Density gradient fractions of EV were tested by anti-mouse IgM ELISA under buffer +0.25% TX100 conditions for 11kG (left) and 110kG (right) EV populations. (C) Density gradient fractions were adsorbed onto aldehyde sulfate microbeads, stained for IgM and CD81, and analyzed by flow cytometry. Dot plots for IgM (top), CD81 (middle), and isotype control (bottom) of fraction 3 are shown, with gates indicating the positive bead populations. The graphs (bottom) show the percentage of positive beads for all 11 kG EV (left) and 110 kG EV (right) fractions. (D) Biparametric dot plots of buffer (left), fraction 3 11 kG EV (center), and fraction 3 110 kG EV (right) samples displaying the expression of isotype control/CD81 or IgM/CD81 are presented. Data presented in (A)–(D) are representative of at least two independent experiments. Data are represented as mean ± SEM.

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Gradient Centrifugation, Expressing, Isolation, Fractionation, Purification, Positive Control, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Control

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: Splenic B cells from WT and μsKO mice release 11kG and 110 kG EV that contain low molecular weight IgM WT mouse splenic B cells were isolated by immunocapture magnetic bead negative selection and cultured unstimulated for 24 h, after which conditioned media was harvested for EV isolation and analysis. (A) Cell surface expression of IgM, CD9, CD63, and CD81 was determined by flow cytometry at the time of plating (day 0) and at harvest (day 1). Histogram overlays of cells stained for antigen (blue) and corresponding FMO stained cells (gray) are presented. (B) EV (11kG and 110kG) in day 1 conditioned media were isolated by differential ultracentrifugation (DUC) and then fractionated by sucrose gradient centrifugation. Following buffer wash, each fraction was resuspended in an equivalent volume and then resolved by SDS/PAGE for immunoblot detection of IgM and CD9, as indicated. A sample of day 1 WT splenic B cell lysate (3.75 μg protein) was run as a positive control. Data are representative of at least two independent experiments. (C) WT and μsKO splenic B cells were isolated by immunocapture magnetic bead negative selection and cultured unstimulated for 24 h, after which conditioned media was collected and cell lysates were prepared. EV were isolated from conditioned media by DUC and resuspended in an equivalent volume of buffer. Equivalent protein (cell lysates; 2 μg) or volume (2kG supernatant, 11 kG EV, 110 kG EV, post-110kG supernatant) for WT and μsKO samples was resolved by SDS/PAGE under unreduced conditions, followed by immunoblot detection of IgM and CD81. (D) The amount of IgM, as determined by anti-mouse IgM ELISA under buffer and buffer +0.25% TX100 conditions, is shown for 2kG supernatant (top), 11 kG EV (lower left), and 110 kG EV (lower right). Data in (C) and (D) are representative of two independent experiments. Data are represented as mean ± SEM. See also Figure S4 .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Molecular Weight, Isolation, Selection, Cell Culture, Expressing, Flow Cytometry, Staining, Gradient Centrifugation, SDS Page, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: EV isolated from WT and μsKO mouse peritoneal cavity fluid and peritoneal B cell cultures contain low molecular weight IgM (A) Peritoneal cavity washout fluid was obtained from WT and μsKO mice and subjected to DUC to isolate 2kG, 11kG, and 110 kG EV populations, which were then resuspended in an equivalent volume of buffer, resolved by SDS/PAGE under unreduced conditions, and immunoblotted for IgM and CD81, along with 2kG and 110kG supernatant samples. Data are representative of two independent experiments. (B) IgM expression in the peritoneal cavity samples described in (A) was determined by anti-mouse IgM ELISA under buffer +0.25% TX100 conditions. Results are shown for the EV samples (left) and 110kG supernatant (right). (C) Peritoneal B-1a cells (CD5 + B220 lo CD43 + CD23 − CD19 + ), peritoneal B2 (B2P) cells (CD5 - B220 high CD43 − CD23 + CD19 + ), and splenic B2 (B2 S) cells (CD5 - B220 high CD43 − CD23 high CD21 lo/− CD19 + ) were sorted from WT and μsKO, as detailed in materials and methods. The sorted B-1a, B2P, and B2 S cells were cultured in media alone or media + LPS (25 μg/mL) for 24-h, after which conditioned media was harvested and the 11kG and 110 kG EV populations were subsequently isolated by differential ultracentrifugation. EV samples (11 kG EV, left and 110 kG EV, right) were resuspended in an equivalent volume of buffer, resolved by SDS/PAGE under non-reducing conditions, and immunoblotted for IgM. Data are representative of at least two independent experiments. See also Figures S5 and .

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Isolation, Molecular Weight, SDS Page, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet: IgM contained in B cell-derived EV can bind antigen CH12 cells were cultured in media alone for two days, after which the conditioned media was subjected to differential ultracentrifugation (DUC) to isolate EV. The EV samples (2kG, 11kG, and 110kG) were resuspended in an equivalent volume of buffer for subsequent analysis by immunoblot and ELISA. (A) An equivalent proportion of each sample was resolved by SDS/PAGE under unreduced conditions, and immunoblotted for IgM and CD81. CH12 cell lysate (lane 1; 7.5 μg) and 110kG supernatant (lane 5), were run as controls. (B) The amount of IgM in each EV sample under buffer +0.25% TX100 conditions was determined by anti-mouse IgM ELISA. For (A) and (B), data are representative of two independent experiments. (C) CH12 cells were incubated with PtC liposomes for 30 min on ice at 1:2000 stock and at sequential 1:2 dilutions thereof, after which FITC fluorescence was determined by flow cytometry and GMI determined. The extreme left data point is the GMI FITC for CH12 cells incubated in buffer only. The turquoise arrowhead indicates the reagent concentration (1:5000 stock) utilized for subsequent experiments. (D) WEHI 231 and CH12 cells were stained with IgM APC and PtC liposome FITC reagent for flow cytometry analysis. Dot plots indicate FMO PtC liposome (first row), FMO IgM APC (second row), and complete stain (third row) fluorescence profiles of single, viable WEHI-231 (left column) and CH12 (right column) cells. (E) EV samples (11kG and 110kG) were isolated by DUC from CH12 conditioned media, resuspended in an equivalent volume of buffer, sequentially titrated 1:2, incubated with an identical volume of aldehyde sulfate microbeads, and subsequently stained with a fluorescent panel of IgM APC, CD81 BV241, CD4 PE/Cy5 and PtC liposome FITC reagent for flow cytometry analysis. GMI for each fluorophore is shown for the most concentrated EV preparation (extreme right data point) and each titrated sample (progressing left). GMI fluorescence of beads incubated in buffer only is indicated by the extreme left data point. Data are representative of two independent experiments. (F) EV (11kG and 110kG) were isolated from WEHI-231 and CH12 conditioned media by differential ultracentrifugation and then titrated 1:2, adsorbed onto an equivalent volume of aldehyde sulfate microbeads, and then stained with the reagent panel described above for flow cytometry analysis (11 kG EV, left graphs; 110kG right graphs). GMI for each fluorophore is shown for the most concentrated EV preparation (extreme right data point) and each titrated sample (progressing left). GMI fluorescence of beads incubated in buffer only is indicated by the extreme left data point. (G) EV (11kG and 110kG) were isolated from CH12 conditioned media by DUC and subjected to sucrose gradient fractionation. Equivalent volumes of resultant fractions were subjected to SDS/PAGE under non-reducing conditions and immunoblotted for IgM and CD81. Data are representative of two independent experiments. (H) Fraction 4 from 11 kG EV and fraction 3 from 110 kG EV were analyzed by flow cytometry following adsorption to aldehyde sulfate microbeads and staining with the reagent panel described above. GMI fluorescence for each fluorophore is shown. GMI fluorescence of beads incubated in buffer only is indicated by the extreme left data point. Data are represented as mean ± SEM.

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Derivative Assay, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Incubation, Liposomes, Fluorescence, Flow Cytometry, Concentration Assay, Staining, Isolation, Fractionation, Adsorption

Journal: iScience

Article Title: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells

doi: 10.1016/j.isci.2023.107526

Figure Lengend Snippet:

Article Snippet: anti-mouse IgM ELISA kit , Bethyl , Cat.# E90-101.

Techniques: Control, Recombinant, Liposomes, Red Blood Cell Lysis, Staining, Lysis, Protease Inhibitor, Purification, Cell Isolation, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Membrane, Microscopy, Flow Cytometry, Knock-Out, Software

Journal: Communications Biology

Article Title: NDRG1 is induced by antigen-receptor signaling but dispensable for B and T cell self-tolerance

doi: 10.1038/s42003-022-04118-w

Figure Lengend Snippet: a Ndrg1 mRNA expression calculated relative to Gapdh expression in Ig HEL primary B lymphocytes stimulated ex vivo with the indicated concentrations of anti-IgM or sHEL, with or without anti-CD40 or LPS for 24 h at 37 °C. b Ndrg1 mRNA expression calculated relative to Gapdh expression in murine A20 Ig HEL tg B cells cultured for 24 h at 37 °C with anti-IgM and with or without LPS. Lines show means with 95% confidence interval (CI) error bars. Data presented are from two to four replicate qPCR readings for each condition and is representative of 3 independent experiments using Ig HEL or non-tg ( a ) or pooled from two A20 ( b ) independent experiments. Significance was determined using a two-way ANOVA with post-hoc Tukey’s testing, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Bethyl laboratories mouse IgG (E90–131), IgM (E90–101) and IgA (E90–103) quantification kits were used by protocol with serum titration of samples as follows: IgG 1:4000, IgM 1:2000, IgA 1:2000, developed using TMB substrate (Life Technologies; 00-4201-56) and detected at 450 nm.

Techniques: Expressing, Ex Vivo, Cell Culture

Journal: Communications Biology

Article Title: NDRG1 is induced by antigen-receptor signaling but dispensable for B and T cell self-tolerance

doi: 10.1038/s42003-022-04118-w

Figure Lengend Snippet: a Representative flow cytometry plots of 50:50 mixtures of WT CD45.1 + Ig HEL and WT or Ndrg1 −/− CD45.2 + Ig HEL splenocytes labeled with CTV dye and transferred by i.v . injection into non-tg or sHEL-tg CD45.2 + recipients for 48 h, showing live B220 + CD19 + gated populations and % transferred CTV + cells. b Percentage of total lymphocytes that were either CD45.1 + or CD45.2 + CTV + as gated in (a), with white points showing paired CD45.1 + Ig HEL cells from donor, colored points are CD45.2 + . c MFI of surface marker expression CD86, CD23, CXCR5, IgM a , BAFF-R and CD95, and intracellular Bim and Bcl2, in transferred Ig HEL B cells, gated as in ( a ). Data points are individual mice and lines show means with 95% CI error bars and data are representative from 1 to 3 independent experiments with 4–6 mice per group. d , e Ratio of B220 + cells surviving from 50:50 mixtures of WT CD45.1 + and WT or Ndrg1 −/− CD45.2 + non-tg B cells stimulated with anti-IgM at the indicated concentrations for 16 h at 37 °C ex vivo ( d ) and CD86 surface expression as MFI on the CD45.2 + B220 + cells ( e ). Data are representative of 3 independent experiments with columns or circles as means with 95% CI error bars.

Article Snippet: Bethyl laboratories mouse IgG (E90–131), IgM (E90–101) and IgA (E90–103) quantification kits were used by protocol with serum titration of samples as follows: IgG 1:4000, IgM 1:2000, IgA 1:2000, developed using TMB substrate (Life Technologies; 00-4201-56) and detected at 450 nm.

Techniques: Flow Cytometry, Labeling, Injection, Marker, Expressing, Ex Vivo

Journal: Cell reports

Article Title: MDA-LDL vaccination induces athero-protective germinal-center-derived antibody responses.

doi: 10.1016/j.celrep.2022.111468

Figure Lengend Snippet: Figure 1. MDA-LDL is athero-protective and promotes a specific humoral response (A) Experimental design. Ldlr/ mice were immunized subcutaneously (s.c.) with MDA-LDL in CFA or with CFA alone (day 0). Four booster immunizations were done given intraperitoneally (i.p.) with MDA-LDL in IFA or IFA alone every two weeks (weeks 2, 4, 6, 8), followed by three additional immunizations once a month four weeks later (week 12, 16, 20). Mice were fed with 1% cholesterol HFD and with 1.25% cholesterol HFD from weeks 8–10 and 10–23, respectively. (B) Representative Oil red O/Hematoxylin staining and (C) quantification of the proportion of atheroma plaque in serial 80 mm-spaced aortic sinus cryosections from MDA-LDL + CFA and CFA immunized mice. (D) Area under the curve (AUC) plot of atheroma plaque quantification. The scale bars in images represents 200 mm. Representative flow cytometry plots and quantification of (E) splenic MBCs (B220+PD-L2+) and (F) IgG1+ bone marrow PCs (B220CD138+IgG1+) from MDA-LDL + CFA and CFA mice. Population percentages in FACS graphs indicate the frequency of live cells. (G) Quantification by ELISA of MDA-LDL specific IgM, IgG1, IgG2b and IgG2c levels in serum from LDLR/ mice 23 weeks after primary immunization with MDA- LDL + CFA or CFA. Data analyzed by unpaired t and two-way ANOVA statistical tests. All experiments were performed with 21–28-week-old males. Each dot in the graphs represents a biological replicate (individual mouse). *p % 0.05, **p < 0.01 and ****p < 0.0001.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for immunoglobulin detection Total, MDA-LDL- andMDA-BSA-specific IgG and IgM antibody titers in plasma or serum were determined using mouse IgM, mouse IgG, mouse IgG1, mouse IgG2b and mouse IgG2c ELISA Quantitation Kits or antibodies (Bethyl laboratories; E90-101, E90–131, A90-105P, A90-109P, A90-136P, respectively) used in accordance with the manufacturer’s instructions.

Techniques: Staining, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: MDA-LDL vaccination induces athero-protective germinal-center-derived antibody responses.

doi: 10.1016/j.celrep.2022.111468

Figure Lengend Snippet: Figure 3. Anti-MDA-LDL antibodies recognize epitopes only presented in the MDA-modified LDL Plasma antibody levels in Aicda-Cre+/ki R26tdTom+/ki mice immunized with CFA alone or MDA-LDL + CFA as determined by ELISA. (A–D) Quantification of (A) IgM, (B) IgG1, (C) IgG2b, and (D) IgG2c anti-MDA-LDL antibodies 2, 4, 6, and 8 weeks after primary immunization. (E) Quantification of IgM and IgG anti-MDA-BSA antibodies 6 weeks after primary immunization. (F) Competition immunoassay to measure anti-MDA-LDL IgG1 antibodies. Plasma IgG1 binding to plated MDA-LDL was quantified in the presence (B) or absence (B0) of increasing concentrations (0, 25, 50, 100, 150, 200, and 300 mg/mL) of the indicated competitors (BSA, MDA-BSA, LDL, and MDA-LDL). Results are expressed as ratios of IgG1 binding to MDA-LDL in the presence (B) or absence (B0) of the competitor. Each dot in the graphs represents a biological replicate (individual mouse). Data analyzed by one-way and two-way ANOVA statistical tests. *p % 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for immunoglobulin detection Total, MDA-LDL- andMDA-BSA-specific IgG and IgM antibody titers in plasma or serum were determined using mouse IgM, mouse IgG, mouse IgG1, mouse IgG2b and mouse IgG2c ELISA Quantitation Kits or antibodies (Bethyl laboratories; E90-101, E90–131, A90-105P, A90-109P, A90-136P, respectively) used in accordance with the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Cell reports

Article Title: MDA-LDL vaccination induces athero-protective germinal-center-derived antibody responses.

doi: 10.1016/j.celrep.2022.111468

Figure Lengend Snippet: Figure 6. Low MDA-LDL-specific antibody responses in the absence of GC-derived PCs (A) Experimental design. BM from Prdm1+/+ Aicda-Cre+/ki or Prdm1fl/flAicda-Cre+/ki mice was i.v. transferred to into irradiated Ldlr/recipients. Four weeks later, mice were immunized with MDA-LDL + CFA, CFA alone, or PBS and fed an HFD with 1.25% cholesterol for 15 weeks. Twenty-three weeks after the primary immunization, mice were sacrificed for analysis. (B) Quantification of SP PCs (B220/lowCD138+) from MDA-LDL + CFA-immunized Prdm1+/+ and Prdm1fl/flAicda-Cre+/ki Ldlr/ chimeras by FACS. (C and D) ELISA-based quantification of total (C) IgG and (D) IgM antibody levels in plasma from Prdm1+/+ and Prdm1fl/flAicda-Cre+/ki Ldlr/ chimeras immunized with MDA-LDL + CFA. (E–H) MDA-LDL-specific (E) IgG1, (F) IgG2b, (G) IgG2c, and (H) IgM antibody levels in Prdm1+/+ and Prdm1fl/flAicda-Cre+/ki Ldlr/ chimeras immunized with MDA-LDL + CFA measured by ELISA. All experiments were performed with 8- to 9-week-old male receptors. Each dot in the graphs represents a biological replicate (individual mouse). Data analyzed by unpaired t statistical test. *p % 0.05, **p < 0.01, and ****p < 0.0001.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for immunoglobulin detection Total, MDA-LDL- andMDA-BSA-specific IgG and IgM antibody titers in plasma or serum were determined using mouse IgM, mouse IgG, mouse IgG1, mouse IgG2b and mouse IgG2c ELISA Quantitation Kits or antibodies (Bethyl laboratories; E90-101, E90–131, A90-105P, A90-109P, A90-136P, respectively) used in accordance with the manufacturer’s instructions.

Techniques: Derivative Assay, Irradiation, Enzyme-linked Immunosorbent Assay, Clinical Proteomics