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Becton Dickinson lsr ii flow cytometer
Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated <t>promastigotes</t> illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD <t>LSR</t> II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P
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1) Product Images from "Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo"

Article Title: Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.01368

Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated promastigotes illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD LSR II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P
Figure Legend Snippet: Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated promastigotes illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD LSR II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P

Techniques Used: TUNEL Assay

2) Product Images from "Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents"

Article Title: Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents

Journal: Microorganisms

doi: 10.3390/microorganisms9050924

Flow cytometry of E. coli challenged with antibiotics. At 0 h, 0.5 h, 2 h, and 5 h time points, viability of E. coli MG1655 challenged with ~1× MBC of ampicillin ( A , B ), ~1× MBC of polymyxin B ( C , D ), ~1× MBC of ciprofloxacin ( E , F ), and ~1× MIC of chloramphenicol ( G , H ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F , H ) and a LSR-II flow cytometer ( A , C , E , G ) and by culture-based enumeration ( B , D , F , H ). FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E , G ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F , H ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.
Figure Legend Snippet: Flow cytometry of E. coli challenged with antibiotics. At 0 h, 0.5 h, 2 h, and 5 h time points, viability of E. coli MG1655 challenged with ~1× MBC of ampicillin ( A , B ), ~1× MBC of polymyxin B ( C , D ), ~1× MBC of ciprofloxacin ( E , F ), and ~1× MIC of chloramphenicol ( G , H ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F , H ) and a LSR-II flow cytometer ( A , C , E , G ) and by culture-based enumeration ( B , D , F , H ). FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E , G ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F , H ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

Techniques Used: Flow Cytometry, Fluorescence, Staining, Generated, Cell Counting

Flow cytometry of untreated E. coli and E. coli challenged with isopropanol. At 0 h and 0.25 h time points, viability of E. coli MG1655 challenged with 70% isopropanol ( C , D ) or nothing (untreated) at 37 °C ( A , B ) and 28 °C ( E , F ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F ) and a LSR-II flow cytometer ( A , C , E ) and by culture-based enumeration ( B , D , F ). Measurements of untreated cells at 37 °C were taken at 0 h, 0.5 h, 2 h, and 5 h time points to reflect the time points for antibiotic challenge, while for untreated cells at 28 °C, measurements were taken at 0 h and 0.25 h to reflect the isopropanol challenge. FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.
Figure Legend Snippet: Flow cytometry of untreated E. coli and E. coli challenged with isopropanol. At 0 h and 0.25 h time points, viability of E. coli MG1655 challenged with 70% isopropanol ( C , D ) or nothing (untreated) at 37 °C ( A , B ) and 28 °C ( E , F ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F ) and a LSR-II flow cytometer ( A , C , E ) and by culture-based enumeration ( B , D , F ). Measurements of untreated cells at 37 °C were taken at 0 h, 0.5 h, 2 h, and 5 h time points to reflect the time points for antibiotic challenge, while for untreated cells at 28 °C, measurements were taken at 0 h and 0.25 h to reflect the isopropanol challenge. FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

Techniques Used: Flow Cytometry, Fluorescence, Staining, Generated, Cell Counting

3) Product Images from "IKK inhibition by BMS-345541 suppresses breast tumorigenesis and metastases by targeting GD2+ cancer stem cells"

Article Title: IKK inhibition by BMS-345541 suppresses breast tumorigenesis and metastases by targeting GD2+ cancer stem cells

Journal: Oncotarget

doi: 10.18632/oncotarget.16294

Activation of NFκB signaling in GD2 + cells A . MDA-MB-231 cells were stained with anti-GD2 conjugated with phycoerythrin (fluorochrome) or Sm152 (metal) and analyzed on an LSR II flow cytometer or by CyTOF, respectively. The data were analyzed on FlowJo software. B . MDA-MB-231 cells were incubated with anti-GD2 antibody conjugated with Sm152, anti-pNFκB(p65) conjugated with Sm149, anti-pPI3K(Y607) antibody conjugated with Gd158, and anti-pmTOR(S248) antibody conjugated with Dy164. All the samples were co-stained with and iridium (DNA-intercalator) conjugated with Rh103 for gating. The cells were analyzed on by CyTOF. The data were analyzed using the SPADE complex data analysis tool. A gradient from green (low) to red (high) indicates expression of each marker in different populations in the SPADE tree structure.. C . qRT-PCR analysis of IKKα in control and IKKα knockdown MDA-MB-231 cells. D . Western blot analysis to determine IKKα and GD3S protein expression in control and IKKα knockdown MDA-MB-231 cells. E . Bar graph represents quantification of IKKα and GD3S protein expression in control and IKKα knockdown MDA-MB-231 cells. GAPDH served as loading control F . Flow cytometry analysis was performed to measure GD2 expression in control and IKKα knockdown MDA-MB-231 cells. G . Bar graph represents the percentage of GD2 + cells in control and IKKα knockdown MDA-MB-231 cells.
Figure Legend Snippet: Activation of NFκB signaling in GD2 + cells A . MDA-MB-231 cells were stained with anti-GD2 conjugated with phycoerythrin (fluorochrome) or Sm152 (metal) and analyzed on an LSR II flow cytometer or by CyTOF, respectively. The data were analyzed on FlowJo software. B . MDA-MB-231 cells were incubated with anti-GD2 antibody conjugated with Sm152, anti-pNFκB(p65) conjugated with Sm149, anti-pPI3K(Y607) antibody conjugated with Gd158, and anti-pmTOR(S248) antibody conjugated with Dy164. All the samples were co-stained with and iridium (DNA-intercalator) conjugated with Rh103 for gating. The cells were analyzed on by CyTOF. The data were analyzed using the SPADE complex data analysis tool. A gradient from green (low) to red (high) indicates expression of each marker in different populations in the SPADE tree structure.. C . qRT-PCR analysis of IKKα in control and IKKα knockdown MDA-MB-231 cells. D . Western blot analysis to determine IKKα and GD3S protein expression in control and IKKα knockdown MDA-MB-231 cells. E . Bar graph represents quantification of IKKα and GD3S protein expression in control and IKKα knockdown MDA-MB-231 cells. GAPDH served as loading control F . Flow cytometry analysis was performed to measure GD2 expression in control and IKKα knockdown MDA-MB-231 cells. G . Bar graph represents the percentage of GD2 + cells in control and IKKα knockdown MDA-MB-231 cells.

Techniques Used: Activation Assay, Multiple Displacement Amplification, Staining, Flow Cytometry, Cytometry, Software, Incubation, Expressing, Marker, Quantitative RT-PCR, Western Blot

4) Product Images from "Myeloid-derived suppressor cell function and epigenetic expression evolves over time after surgical sepsis"

Article Title: Myeloid-derived suppressor cell function and epigenetic expression evolves over time after surgical sepsis

Journal: Critical Care

doi: 10.1186/s13054-019-2628-x

Phenotypic classification of MDSCs. Gating strategy used to classify CD33 + CD11b + HLA-DR −/low MDSC populations in the human whole blood using samples stained according to the protocol and analyzed on the LSR II flow cytometer. Monocytic MDSC subpopulations were further characterized as CD14 + and granulocytic MDSCs as CD14 − CD15 +
Figure Legend Snippet: Phenotypic classification of MDSCs. Gating strategy used to classify CD33 + CD11b + HLA-DR −/low MDSC populations in the human whole blood using samples stained according to the protocol and analyzed on the LSR II flow cytometer. Monocytic MDSC subpopulations were further characterized as CD14 + and granulocytic MDSCs as CD14 − CD15 +

Techniques Used: Staining, Flow Cytometry, Cytometry

5) Product Images from "Vaccination with Ad5 Vectors Expands Ad5-Specific CD8+ T Cells without Altering Memory Phenotype or Functionality"

Article Title: Vaccination with Ad5 Vectors Expands Ad5-Specific CD8+ T Cells without Altering Memory Phenotype or Functionality

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014385

Baseline CD8 + T-cell responses. Forty total subjects with a range of Ad5 nAb titers were analyzed. Five seronegative (Ad5 nAb titer ≤18, gray symbols) and five seropositive subjects (Ad5 nAb titer > 18, black symbols and white boxes) received Merck Ad5 gag/pol/nef as described in Methods . Each circle represents a subject with lines representing the mean. CD8 + T-cell responses were measured by flow cytometry following whole Ad5 vector stimulation. A ) Gating strategy for measuring Ad5-specific T cells by intracellular cytokine staining. At least 100,000 PBMCs were collected on a modified LSR II. Singlets were selected with a FSC-A and FSC-H, followed by a lymphocytes gate, dead cell exclusion, and exclusion of contaminating CD14 + monocytes and CD19 + B-cells. CD3 + T-cells were selected and then CD8 + cells by CD8 + CD4 − . Central memory, effector memory and effector CD8 + T cells were selected before gating on each cytokine. Because cells can store perforin and these appear perforin + , Ad5-specific CD8 + perforin + T cells must also be positive for another function to be considered as a responding event. B ) Total Ad-specific CD8 + response. The total Ad-specific CD8 + response was computed by summing cells making at least IL-2, IFN-γ, MIP1α, or TNFα as measured by flow cytometry. C ) There was no difference in the magnitude of the Ad-specific CD8 + T-cell response between serogroups at baseline. D ) There was no correlation between the magnitude of Ad-specific CD8 + T-cell responses and nAb titer at baseline. E ) There is a correlation between the magnitude of Ad-specific CD4 + and Ad-specific CD8 + T-cell responses at baseline.
Figure Legend Snippet: Baseline CD8 + T-cell responses. Forty total subjects with a range of Ad5 nAb titers were analyzed. Five seronegative (Ad5 nAb titer ≤18, gray symbols) and five seropositive subjects (Ad5 nAb titer > 18, black symbols and white boxes) received Merck Ad5 gag/pol/nef as described in Methods . Each circle represents a subject with lines representing the mean. CD8 + T-cell responses were measured by flow cytometry following whole Ad5 vector stimulation. A ) Gating strategy for measuring Ad5-specific T cells by intracellular cytokine staining. At least 100,000 PBMCs were collected on a modified LSR II. Singlets were selected with a FSC-A and FSC-H, followed by a lymphocytes gate, dead cell exclusion, and exclusion of contaminating CD14 + monocytes and CD19 + B-cells. CD3 + T-cells were selected and then CD8 + cells by CD8 + CD4 − . Central memory, effector memory and effector CD8 + T cells were selected before gating on each cytokine. Because cells can store perforin and these appear perforin + , Ad5-specific CD8 + perforin + T cells must also be positive for another function to be considered as a responding event. B ) Total Ad-specific CD8 + response. The total Ad-specific CD8 + response was computed by summing cells making at least IL-2, IFN-γ, MIP1α, or TNFα as measured by flow cytometry. C ) There was no difference in the magnitude of the Ad-specific CD8 + T-cell response between serogroups at baseline. D ) There was no correlation between the magnitude of Ad-specific CD8 + T-cell responses and nAb titer at baseline. E ) There is a correlation between the magnitude of Ad-specific CD4 + and Ad-specific CD8 + T-cell responses at baseline.

Techniques Used: Flow Cytometry, Cytometry, Plasmid Preparation, Staining, Modification

6) Product Images from "Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition"

Article Title: Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition

Journal: Cellular microbiology

doi: 10.1111/j.1462-5822.2009.01385.x

Wild type Ebola GP mediates entry into human DCs (A) Protein equivalents of purified VP40+GP (lane 2), lacVP40 (lane 3), lacVP40+GP (lane 4), lacVP40+GP L561A mutant (lane 5) or lacVP40+GP F88A (lane 6) VLPs were lysed and tested for total β-lactamase activity using a fluorogenic substrate (Lytic Blazer, Invitrogen, Carlsbad, CA). The y-axis denotes the RFU (relative fluorescence units) × 1000. (B and C) Mock (histogram 1) and lactamase equivalents of VP40+GP (histogram 2 and second dotplot), lacVP40 (histogram 3 and third dotplot), lacVP40+GP (histogram 4 and fourth dotplot), lacVP40+GP (L561A) (histogram 5 and fifth dotplot) and lacVP40+GP (F88A) (histogram 6 and sixth dotplot) VLPs were incubated with human DCs for 3 hours to allow infection after which DCs were loaded with fluorogenic CCF2-AM substrate. Ebola VLP entry was assayed by measuring the amount of cytoplasmic substrate (fluoresces ~520 nm) that was β-lactamase-cleaved into its product (fluoresces ~460nm) using a (B) FLUOstar OPTIMA plate reader (expressed as a ratio) and (C) using an LSR II flow cytometer (data expressed as a percentage of cells that have an increased blue fluorescence and decreased green fluorescence). Entry was detected only with the VLPs produced with lacVP40 and wildtype GP (fourth panel).
Figure Legend Snippet: Wild type Ebola GP mediates entry into human DCs (A) Protein equivalents of purified VP40+GP (lane 2), lacVP40 (lane 3), lacVP40+GP (lane 4), lacVP40+GP L561A mutant (lane 5) or lacVP40+GP F88A (lane 6) VLPs were lysed and tested for total β-lactamase activity using a fluorogenic substrate (Lytic Blazer, Invitrogen, Carlsbad, CA). The y-axis denotes the RFU (relative fluorescence units) × 1000. (B and C) Mock (histogram 1) and lactamase equivalents of VP40+GP (histogram 2 and second dotplot), lacVP40 (histogram 3 and third dotplot), lacVP40+GP (histogram 4 and fourth dotplot), lacVP40+GP (L561A) (histogram 5 and fifth dotplot) and lacVP40+GP (F88A) (histogram 6 and sixth dotplot) VLPs were incubated with human DCs for 3 hours to allow infection after which DCs were loaded with fluorogenic CCF2-AM substrate. Ebola VLP entry was assayed by measuring the amount of cytoplasmic substrate (fluoresces ~520 nm) that was β-lactamase-cleaved into its product (fluoresces ~460nm) using a (B) FLUOstar OPTIMA plate reader (expressed as a ratio) and (C) using an LSR II flow cytometer (data expressed as a percentage of cells that have an increased blue fluorescence and decreased green fluorescence). Entry was detected only with the VLPs produced with lacVP40 and wildtype GP (fourth panel).

Techniques Used: Purification, Mutagenesis, Activity Assay, Fluorescence, Incubation, Infection, Flow Cytometry, Cytometry, Produced

7) Product Images from "Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo"

Article Title: Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.01368

Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated promastigotes illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD LSR II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P
Figure Legend Snippet: Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated promastigotes illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD LSR II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P

Techniques Used: TUNEL Assay

8) Product Images from "Regulatory T cells Exhibit Decreased Proliferation but Enhanced Suppression After Pulsing with Sirolimus"

Article Title: Regulatory T cells Exhibit Decreased Proliferation but Enhanced Suppression After Pulsing with Sirolimus

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2011.03963.x

The enhanced function of SPTs is accompanied by alterations in their proliferative capacity and phenotype (A): SPTs are less proliferative than unpulsed Tregs in an allo-MLR. SPTs and control Tregs were stained with CTV dye and were added to an MLR in which effector T cells were stained with CFSE. Suppression of effector T cell allo-proliferation (not shown) and proliferation of SPTs and control Tregs were determined. Shown is a representative tracing of proliferation as measured by the dilution of CTV fluorescence, comparing unpulsed Tregs to SPTs. (B): Summary of the relative proliferation of SPTs compared to control Tregs from three independent experiments. Shown is the mean +/− SEM. (C): Both dividing and non-dividing cells from the ex vivo expanded Treg cultures express FoxP3: SPTs were labeled with CTV and placed into the MLR. FoxP3 fluorescence was measured on the proliferating SPTs (CTV low , green traces) and non-proliferating SPTs (CTV high , purple traces) as well as on the non-Tregs (black traces). (D). SPTs exhibit higher cell-surface expression of CD25 compared to unpulsed Tregs. SPTs and control, unpulsed Tregs were stained for CD3, CD4 CD25 and intracellular FoxP3 and flow cytometry data was acquired on BD LSR II and analyzed by Flowjo. A representative histogram (left) and summary expression data (displayed as the mean fluorescence intensity (MFI) from five independent experiments (right) for CD25 fluorescence intensity is shown. Shown is the mean +/− SEM. (E) SPT cultures have more CTLA-4 High Tregs compared to control, unpulsed Treg cultures. SPTs and control unpulsed Tregs were stained for CD3, CD4, total CTLA-4 and intracellular FoxP3, and flow cytometry data was acquired on BD LSR II and analyzed by Flowjo. Left: Representative density plots showing the number of Tregs in each culture that was CTLA-4 High Right: Summary data of CTLA-4 High expression from four independent experiments is shown. Shown is the mean +/− SEM.
Figure Legend Snippet: The enhanced function of SPTs is accompanied by alterations in their proliferative capacity and phenotype (A): SPTs are less proliferative than unpulsed Tregs in an allo-MLR. SPTs and control Tregs were stained with CTV dye and were added to an MLR in which effector T cells were stained with CFSE. Suppression of effector T cell allo-proliferation (not shown) and proliferation of SPTs and control Tregs were determined. Shown is a representative tracing of proliferation as measured by the dilution of CTV fluorescence, comparing unpulsed Tregs to SPTs. (B): Summary of the relative proliferation of SPTs compared to control Tregs from three independent experiments. Shown is the mean +/− SEM. (C): Both dividing and non-dividing cells from the ex vivo expanded Treg cultures express FoxP3: SPTs were labeled with CTV and placed into the MLR. FoxP3 fluorescence was measured on the proliferating SPTs (CTV low , green traces) and non-proliferating SPTs (CTV high , purple traces) as well as on the non-Tregs (black traces). (D). SPTs exhibit higher cell-surface expression of CD25 compared to unpulsed Tregs. SPTs and control, unpulsed Tregs were stained for CD3, CD4 CD25 and intracellular FoxP3 and flow cytometry data was acquired on BD LSR II and analyzed by Flowjo. A representative histogram (left) and summary expression data (displayed as the mean fluorescence intensity (MFI) from five independent experiments (right) for CD25 fluorescence intensity is shown. Shown is the mean +/− SEM. (E) SPT cultures have more CTLA-4 High Tregs compared to control, unpulsed Treg cultures. SPTs and control unpulsed Tregs were stained for CD3, CD4, total CTLA-4 and intracellular FoxP3, and flow cytometry data was acquired on BD LSR II and analyzed by Flowjo. Left: Representative density plots showing the number of Tregs in each culture that was CTLA-4 High Right: Summary data of CTLA-4 High expression from four independent experiments is shown. Shown is the mean +/− SEM.

Techniques Used: Staining, Fluorescence, Ex Vivo, Labeling, Expressing, Flow Cytometry, Cytometry, Single-particle Tracking

9) Product Images from "Improved NYVAC-Based Vaccine Vectors"

Article Title: Improved NYVAC-Based Vaccine Vectors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025674

Comparative analysis of the apoptosis induction of non-replication versus replication-competent B19R deletion mutants. The different stages of cell cycle and the percentage of cells with subG 0 DNA content were analyzed by propidium iodide (PI) staining and FACS analysis. HeLa cells were mock-infected or infected at an MOI of 5 with NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At 24 hours post-infection cells were harvested and processed as described in Materials and Methods . The percentage of cells with hypodiploid DNA content was determined using an LSR II flow cytometer (Becton Dickinson). The results are expressed as fold increase in apoptotic cells with respect to uninfected cells.
Figure Legend Snippet: Comparative analysis of the apoptosis induction of non-replication versus replication-competent B19R deletion mutants. The different stages of cell cycle and the percentage of cells with subG 0 DNA content were analyzed by propidium iodide (PI) staining and FACS analysis. HeLa cells were mock-infected or infected at an MOI of 5 with NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At 24 hours post-infection cells were harvested and processed as described in Materials and Methods . The percentage of cells with hypodiploid DNA content was determined using an LSR II flow cytometer (Becton Dickinson). The results are expressed as fold increase in apoptotic cells with respect to uninfected cells.

Techniques Used: Staining, FACS, Infection, Flow Cytometry, Cytometry

10) Product Images from "Attenuation of antigen-specific T helper 1 immunity by Neolitsea hiiranensis and its derived terpenoids"

Article Title: Attenuation of antigen-specific T helper 1 immunity by Neolitsea hiiranensis and its derived terpenoids

Journal: PeerJ

doi: 10.7717/peerj.2758

The effects of β -caryophyllene oxide on protein levels of T-bet and GATA-3 in CD4 + cells. After double staining of CD4 with T-bet or GATA-3 antibodies, tens of thousands of CD4 + cells were acquired on a BD LSR II flow cytometer. (A and C) The representative flow graphs show the cell population of T-bet, GATA-3 or CD4 cells and CD4 + T-bet + and CD4 + GATA-3 + double positive cells. The proportion of CD4 + T-bet + and CD4 + GATA-3 + in total CD4 + cells was quantified. (B and D) The mean fluorescence intensity (MFI) of T-bet or GATA-3 in total CD4 + cells was showed. The results are means ± SE of three separate experiments. * p
Figure Legend Snippet: The effects of β -caryophyllene oxide on protein levels of T-bet and GATA-3 in CD4 + cells. After double staining of CD4 with T-bet or GATA-3 antibodies, tens of thousands of CD4 + cells were acquired on a BD LSR II flow cytometer. (A and C) The representative flow graphs show the cell population of T-bet, GATA-3 or CD4 cells and CD4 + T-bet + and CD4 + GATA-3 + double positive cells. The proportion of CD4 + T-bet + and CD4 + GATA-3 + in total CD4 + cells was quantified. (B and D) The mean fluorescence intensity (MFI) of T-bet or GATA-3 in total CD4 + cells was showed. The results are means ± SE of three separate experiments. * p

Techniques Used: Double Staining, Flow Cytometry, Cytometry, Fluorescence

11) Product Images from "Attenuation of antigen-specific T helper 1 immunity by Neolitsea hiiranensis and its derived terpenoids"

Article Title: Attenuation of antigen-specific T helper 1 immunity by Neolitsea hiiranensis and its derived terpenoids

Journal: PeerJ

doi: 10.7717/peerj.2758

The effects of β -caryophyllene oxide on protein levels of T-bet and GATA-3 in CD4 + cells. After double staining of CD4 with T-bet or GATA-3 antibodies, tens of thousands of CD4 + cells were acquired on a BD LSR II flow cytometer. (A and C) The representative flow graphs show the cell population of T-bet, GATA-3 or CD4 cells and CD4 + T-bet + and CD4 + GATA-3 + double positive cells. The proportion of CD4 + T-bet + and CD4 + GATA-3 + in total CD4 + cells was quantified. (B and D) The mean fluorescence intensity (MFI) of T-bet or GATA-3 in total CD4 + cells was showed. The results are means ± SE of three separate experiments. * p
Figure Legend Snippet: The effects of β -caryophyllene oxide on protein levels of T-bet and GATA-3 in CD4 + cells. After double staining of CD4 with T-bet or GATA-3 antibodies, tens of thousands of CD4 + cells were acquired on a BD LSR II flow cytometer. (A and C) The representative flow graphs show the cell population of T-bet, GATA-3 or CD4 cells and CD4 + T-bet + and CD4 + GATA-3 + double positive cells. The proportion of CD4 + T-bet + and CD4 + GATA-3 + in total CD4 + cells was quantified. (B and D) The mean fluorescence intensity (MFI) of T-bet or GATA-3 in total CD4 + cells was showed. The results are means ± SE of three separate experiments. * p

Techniques Used: Double Staining, Flow Cytometry, Cytometry, Fluorescence

12) Product Images from "Improved NYVAC-Based Vaccine Vectors"

Article Title: Improved NYVAC-Based Vaccine Vectors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025674

Signal transduction. A ) Cells were infected at an MOI of 5; lysates were prepared at 6 hours post-infection and proteins were identified by a standard Western blot probed with anti-gp120 or anti-gag. B ) HeLa cells were infected with the indicated viruses and harvested at 6 hours post-infection. Lysates were prepared and analyzed by Western blot to detect phosphorylation of shown signal transduction pathway components. C ) Human keratinocytes were infected with the indicated viruses and harvested 6 hours post-infection. Lysates were prepared and analyzed by Western blot to detect phosphorylation of shown signal transduction pathway components. D) Comparative analysis of P-STAT1 protein levels of non-replication vs. replication competent B19R deletion mutants. Phospho-STAT1 levels present in extracts of cells infected with different recombinant viruses were measured by Cytometric Bead Array (CBA) using an LSR II flow cytometer. The concentrations of total and phospho STAT1 were measured simultaneously using the Cell Signaling Master Buffer Kit (BD Biosciences Pharmingen). For this CBA analysis, PBMCs were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At 4 hours post-infection, cells were harvested and processed for CBA analysis as described in Materials and Methods and according to manufacturer's recommendations. Results are given as the ratio between phospho and total STAT1. E) Microarray. RNA was extracted from human pDCs infected with the indicated viruses. Data from a subset of the IFN-induced genes is shown. Fold increase (decrease) is normalized to results for mock-infected cells.
Figure Legend Snippet: Signal transduction. A ) Cells were infected at an MOI of 5; lysates were prepared at 6 hours post-infection and proteins were identified by a standard Western blot probed with anti-gp120 or anti-gag. B ) HeLa cells were infected with the indicated viruses and harvested at 6 hours post-infection. Lysates were prepared and analyzed by Western blot to detect phosphorylation of shown signal transduction pathway components. C ) Human keratinocytes were infected with the indicated viruses and harvested 6 hours post-infection. Lysates were prepared and analyzed by Western blot to detect phosphorylation of shown signal transduction pathway components. D) Comparative analysis of P-STAT1 protein levels of non-replication vs. replication competent B19R deletion mutants. Phospho-STAT1 levels present in extracts of cells infected with different recombinant viruses were measured by Cytometric Bead Array (CBA) using an LSR II flow cytometer. The concentrations of total and phospho STAT1 were measured simultaneously using the Cell Signaling Master Buffer Kit (BD Biosciences Pharmingen). For this CBA analysis, PBMCs were mock-infected or infected at an MOI of 5 with NYVAC wt, NYVAC-C, NYVAC-C-KC, NYVAC-C-ΔB19R or NYVAC-C-KC-ΔB19R. At 4 hours post-infection, cells were harvested and processed for CBA analysis as described in Materials and Methods and according to manufacturer's recommendations. Results are given as the ratio between phospho and total STAT1. E) Microarray. RNA was extracted from human pDCs infected with the indicated viruses. Data from a subset of the IFN-induced genes is shown. Fold increase (decrease) is normalized to results for mock-infected cells.

Techniques Used: Transduction, Infection, Western Blot, Recombinant, Crocin Bleaching Assay, Flow Cytometry, Cytometry, Microarray

13) Product Images from "The proportion of CD16+CD14dim monocytes increases with tumor cell load in bone marrow of patients with multiple myeloma"

Article Title: The proportion of CD16+CD14dim monocytes increases with tumor cell load in bone marrow of patients with multiple myeloma

Journal: Immunity, Inflammation and Disease

doi: 10.1002/iid3.53

CD16 + CD14 dim monocytes increase in the bone marrow of myeloma patients. (A) Gating strategy: Cells were stained with a cocktail of antibodies against CD66b, lineage (CD3, CD19, CD56, CD138, CD15, CD34, and CD235a), CD45, HLADR, CD16, and CD14 and analyzed on an LSR II Flow cytometer. Gates were set on FSC and SSC (i), doublets were excluded, and gates were set on the CD45 + cells (ii). Lineage and CD66b + were further excluded (iii). HLA DR + cells are shown in (iv), and CD14 and CD16 profiles as shown in (B) were analyzed on these cells. (B) Representative CD14 and CD16 profiles from bone marrow aspirate from two patients with 30 and 2% PC, respectively. The gates indicated show the populations of % CD16 + CD14 dim and CD14 high cells, respectively. (C) Correlation between the ratio of CD16 + CD14 dim cells/CD14 high cells and percent PC in the bone marrow of patients ( n = 33). Each dot represents a value from a patient as estimated by the gating indicated in Figure 1B . The P -value was calculated from Spearman's test. (D) CD16 + CD14 dim monocytes increase in the bone marrow of patients with intermediate levels of 10–30% plasma cells. Bone marrow monocytes were stained and analyzed as described above and in Methods. Figures show monocyte populations as percent of CD45 + cells. Patients were grouped as low percent PC (0–10%) ( n = 9), intermediate (10–30%) ( n = 12), and high ( > 30%) ( n = 12). Each dot represents a patient. Monocyte populations: (i) CD16 + CD14 dim , (ii) CD14 high , and (iii) all monocyte populations (lineage − granulocyte − CD14 + ). Statistical analysis was performed with Mann–Whitney Test. (E) CD16 + CD14 dim cells express markers defining the CX 3 CR 1 subpopulation of monocytes. Representative histograms of CX 3 CR 1 , CCR2, CD163, and CD62L expression on gated CD16 + CD14 dim and CD14 high monocytes. Gates were set as described in (A). FMO (fluorescence minus one) is used as negative control.
Figure Legend Snippet: CD16 + CD14 dim monocytes increase in the bone marrow of myeloma patients. (A) Gating strategy: Cells were stained with a cocktail of antibodies against CD66b, lineage (CD3, CD19, CD56, CD138, CD15, CD34, and CD235a), CD45, HLADR, CD16, and CD14 and analyzed on an LSR II Flow cytometer. Gates were set on FSC and SSC (i), doublets were excluded, and gates were set on the CD45 + cells (ii). Lineage and CD66b + were further excluded (iii). HLA DR + cells are shown in (iv), and CD14 and CD16 profiles as shown in (B) were analyzed on these cells. (B) Representative CD14 and CD16 profiles from bone marrow aspirate from two patients with 30 and 2% PC, respectively. The gates indicated show the populations of % CD16 + CD14 dim and CD14 high cells, respectively. (C) Correlation between the ratio of CD16 + CD14 dim cells/CD14 high cells and percent PC in the bone marrow of patients ( n = 33). Each dot represents a value from a patient as estimated by the gating indicated in Figure 1B . The P -value was calculated from Spearman's test. (D) CD16 + CD14 dim monocytes increase in the bone marrow of patients with intermediate levels of 10–30% plasma cells. Bone marrow monocytes were stained and analyzed as described above and in Methods. Figures show monocyte populations as percent of CD45 + cells. Patients were grouped as low percent PC (0–10%) ( n = 9), intermediate (10–30%) ( n = 12), and high ( > 30%) ( n = 12). Each dot represents a patient. Monocyte populations: (i) CD16 + CD14 dim , (ii) CD14 high , and (iii) all monocyte populations (lineage − granulocyte − CD14 + ). Statistical analysis was performed with Mann–Whitney Test. (E) CD16 + CD14 dim cells express markers defining the CX 3 CR 1 subpopulation of monocytes. Representative histograms of CX 3 CR 1 , CCR2, CD163, and CD62L expression on gated CD16 + CD14 dim and CD14 high monocytes. Gates were set as described in (A). FMO (fluorescence minus one) is used as negative control.

Techniques Used: Staining, Flow Cytometry, Cytometry, MANN-WHITNEY, Expressing, Fluorescence, Negative Control

14) Product Images from "Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents"

Article Title: Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents

Journal: Microorganisms

doi: 10.3390/microorganisms9050924

Flow cytometry of E. coli challenged with antibiotics. At 0 h, 0.5 h, 2 h, and 5 h time points, viability of E. coli MG1655 challenged with ~1× MBC of ampicillin ( A , B ), ~1× MBC of polymyxin B ( C , D ), ~1× MBC of ciprofloxacin ( E , F ), and ~1× MIC of chloramphenicol ( G , H ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F , H ) and a LSR-II flow cytometer ( A , C , E , G ) and by culture-based enumeration ( B , D , F , H ). FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E , G ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F , H ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.
Figure Legend Snippet: Flow cytometry of E. coli challenged with antibiotics. At 0 h, 0.5 h, 2 h, and 5 h time points, viability of E. coli MG1655 challenged with ~1× MBC of ampicillin ( A , B ), ~1× MBC of polymyxin B ( C , D ), ~1× MBC of ciprofloxacin ( E , F ), and ~1× MIC of chloramphenicol ( G , H ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F , H ) and a LSR-II flow cytometer ( A , C , E , G ) and by culture-based enumeration ( B , D , F , H ). FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E , G ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F , H ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

Techniques Used: Flow Cytometry, Fluorescence, Staining, Generated, Cell Counting

Flow cytometry of untreated E. coli and E. coli challenged with isopropanol. At 0 h and 0.25 h time points, viability of E. coli MG1655 challenged with 70% isopropanol ( C , D ) or nothing (untreated) at 37 °C ( A , B ) and 28 °C ( E , F ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F ) and a LSR-II flow cytometer ( A , C , E ) and by culture-based enumeration ( B , D , F ). Measurements of untreated cells at 37 °C were taken at 0 h, 0.5 h, 2 h, and 5 h time points to reflect the time points for antibiotic challenge, while for untreated cells at 28 °C, measurements were taken at 0 h and 0.25 h to reflect the isopropanol challenge. FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.
Figure Legend Snippet: Flow cytometry of untreated E. coli and E. coli challenged with isopropanol. At 0 h and 0.25 h time points, viability of E. coli MG1655 challenged with 70% isopropanol ( C , D ) or nothing (untreated) at 37 °C ( A , B ) and 28 °C ( E , F ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F ) and a LSR-II flow cytometer ( A , C , E ) and by culture-based enumeration ( B , D , F ). Measurements of untreated cells at 37 °C were taken at 0 h, 0.5 h, 2 h, and 5 h time points to reflect the time points for antibiotic challenge, while for untreated cells at 28 °C, measurements were taken at 0 h and 0.25 h to reflect the isopropanol challenge. FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

Techniques Used: Flow Cytometry, Fluorescence, Staining, Generated, Cell Counting

15) Product Images from "Pivotal Advance: Eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production"

Article Title: Pivotal Advance: Eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production

Journal: Journal of leukocyte biology

doi: 10.1189/jlb.0607392

Eosinophils are required for in vivo alum-elicited, early priming of MHC II-mediated calcium mobilization responses in splenic B cells. Calcium mobilization was evaluated in WT mice (A), following i.p. alum administration (B), in eosinophil-deficient ΔdblGATA mice (C), and in ΔdblGATA mice that received adoptive reconstitutions with eosinophils by i.v. transfers on days 0 and 3 following i.p. alum administration (D). MHC II cross-linking elicited B cell calcium mobilization only in WT mice (B) and in eosinophil-reconstituted ΔdblGATA mice (D) following i.p. alum administration. Single-cell suspensions were prepared from spleens of the indicated mice 6 days after injection of i.p. alum, i.p. alum and i.v. eosinophils, or nothing. Cells were incubated in IMDM for 45 min at 37°C in the presence of Indo 1 AM, 2.4G2 FcR-blocking mAb, anti-B220-PE mAb, and biotinylated anti-MHC class II mAb. MHC class II-mediated calcium mobilization in splenic B220 + cells was triggered by avidin cross-linking and monitored by B220 + -gated flow cytometry (Becton Dickinson LSR II). Ionomycin was a positive control. Calcium mobilization responses in B220 + B cells were analyzed by Becton Dickinson FACSDiva (left panel) or FlowJo software (right panel). Results are representative of one of three experiments.
Figure Legend Snippet: Eosinophils are required for in vivo alum-elicited, early priming of MHC II-mediated calcium mobilization responses in splenic B cells. Calcium mobilization was evaluated in WT mice (A), following i.p. alum administration (B), in eosinophil-deficient ΔdblGATA mice (C), and in ΔdblGATA mice that received adoptive reconstitutions with eosinophils by i.v. transfers on days 0 and 3 following i.p. alum administration (D). MHC II cross-linking elicited B cell calcium mobilization only in WT mice (B) and in eosinophil-reconstituted ΔdblGATA mice (D) following i.p. alum administration. Single-cell suspensions were prepared from spleens of the indicated mice 6 days after injection of i.p. alum, i.p. alum and i.v. eosinophils, or nothing. Cells were incubated in IMDM for 45 min at 37°C in the presence of Indo 1 AM, 2.4G2 FcR-blocking mAb, anti-B220-PE mAb, and biotinylated anti-MHC class II mAb. MHC class II-mediated calcium mobilization in splenic B220 + cells was triggered by avidin cross-linking and monitored by B220 + -gated flow cytometry (Becton Dickinson LSR II). Ionomycin was a positive control. Calcium mobilization responses in B220 + B cells were analyzed by Becton Dickinson FACSDiva (left panel) or FlowJo software (right panel). Results are representative of one of three experiments.

Techniques Used: In Vivo, Mouse Assay, Injection, Incubation, Blocking Assay, Avidin-Biotin Assay, Flow Cytometry, Cytometry, Positive Control, Software

16) Product Images from "Pivotal Advance: Eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production"

Article Title: Pivotal Advance: Eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production

Journal: Journal of leukocyte biology

doi: 10.1189/jlb.0607392

Eosinophils are required for in vivo alum-elicited, early priming of MHC II-mediated calcium mobilization responses in splenic B cells. Calcium mobilization was evaluated in WT mice (A), following i.p. alum administration (B), in eosinophil-deficient ΔdblGATA mice (C), and in ΔdblGATA mice that received adoptive reconstitutions with eosinophils by i.v. transfers on days 0 and 3 following i.p. alum administration (D). MHC II cross-linking elicited B cell calcium mobilization only in WT mice (B) and in eosinophil-reconstituted ΔdblGATA mice (D) following i.p. alum administration. Single-cell suspensions were prepared from spleens of the indicated mice 6 days after injection of i.p. alum, i.p. alum and i.v. eosinophils, or nothing. Cells were incubated in IMDM for 45 min at 37°C in the presence of Indo 1 AM, 2.4G2 FcR-blocking mAb, anti-B220-PE mAb, and biotinylated anti-MHC class II mAb. MHC class II-mediated calcium mobilization in splenic B220 + cells was triggered by avidin cross-linking and monitored by B220 + -gated flow cytometry (Becton Dickinson LSR II). Ionomycin was a positive control. Calcium mobilization responses in B220 + B cells were analyzed by Becton Dickinson FACSDiva (left panel) or FlowJo software (right panel). Results are representative of one of three experiments.
Figure Legend Snippet: Eosinophils are required for in vivo alum-elicited, early priming of MHC II-mediated calcium mobilization responses in splenic B cells. Calcium mobilization was evaluated in WT mice (A), following i.p. alum administration (B), in eosinophil-deficient ΔdblGATA mice (C), and in ΔdblGATA mice that received adoptive reconstitutions with eosinophils by i.v. transfers on days 0 and 3 following i.p. alum administration (D). MHC II cross-linking elicited B cell calcium mobilization only in WT mice (B) and in eosinophil-reconstituted ΔdblGATA mice (D) following i.p. alum administration. Single-cell suspensions were prepared from spleens of the indicated mice 6 days after injection of i.p. alum, i.p. alum and i.v. eosinophils, or nothing. Cells were incubated in IMDM for 45 min at 37°C in the presence of Indo 1 AM, 2.4G2 FcR-blocking mAb, anti-B220-PE mAb, and biotinylated anti-MHC class II mAb. MHC class II-mediated calcium mobilization in splenic B220 + cells was triggered by avidin cross-linking and monitored by B220 + -gated flow cytometry (Becton Dickinson LSR II). Ionomycin was a positive control. Calcium mobilization responses in B220 + B cells were analyzed by Becton Dickinson FACSDiva (left panel) or FlowJo software (right panel). Results are representative of one of three experiments.

Techniques Used: In Vivo, Mouse Assay, Injection, Incubation, Blocking Assay, Avidin-Biotin Assay, Flow Cytometry, Cytometry, Positive Control, Software

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Cytometry:

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Fluorescence:

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    Becton Dickinson lsr ii flow cytometer
    Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated <t>promastigotes</t> illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD <t>LSR</t> II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P
    Lsr Ii Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Assessment of autophagy. ( A ) Histogram of <t>flow</t> cytometric analysis of mutant cybrids (III-8.48) and control cybrids (C59.32) using CYTOID® Autophagy Detection Kit. Three control and three mutant cybrids were incubated with DMEM in the absence and presence of rapamycin (inducers of autophagy) and chloroquine (lysosomal inhibitor) at 37°C for 18 h, added to CYTO-ID ® -Green dye and analyzed using a Novocyte flow <t>cytometer</t> (ACEA Biosciences). ( B ) Relative ratios of Cyto-ID fluorescence intensity from three mutant and three control cell lines. Three independent determinations were done in each cell line. ( C ) Western blot analysis for autophagic response proteins: <t>LC3-I/II</t> and p62. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β -actin as a loading control. ( D ) Quantification of autophagy markers LC3 I/II and p62 in mutant and control cell lines were determined as described elsewhere ( 70 ). Three independent determinations were done in each cell line. G. Graph details and symbols are explained in the legend to Figure 4 .
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    Sirt1 dampens the lupus autoantibody response. ( A ) IgG1 + and IgG2a + ANAs in sera from 35-week-old female Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice (immunofluorescence microscopy of one representative of three independent experiments). Scale bar, 50 μm. ( B ) Anti-dsDNA, anti-histone, anti-RNP, and anti-RNA IgG titers (ELISAs) in sera from 35-week-old female Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice (means ± SD of six Aicda cre Sirt1 +/+ and six Aicda cre Sirt1 fl/fl mice, each tested in triplicates). ( C ) Expression of Sirt1 and Aicda (qRT-PCR analysis) in spleen B cells from C57BL/6 mice and lupus-prone MRL/ Fas lpr/lpr and BXD2 mice. Data are relative expression to β -Actin (means ± SD of three mice in each group). Dotted lines depict trends. ( D ) qRT-PCR analysis of expression of SIRT1 and AICDA in B cells from healthy humans and patients with SLE. Data are normalized to expression of β -ACTIN (means ± SD of three healthy individuals and three patients with SLE). Dotted lines depict trends. ( E ) Acetylated H3K9/K14 in the Aicda, Prdm1 , and Xbp1 promoters (ChIP-qPCR analysis) in B cells from C57BL/6 and MRL/ Fas lpr/lpr mice. Data are ratios of acetylated H3K9/K14 in MRL/ Fas lpr/lpr mice to that in C57BL/6 mice (set as 1; means ± SD of three mice in each group). ( F ) Acetylated H3K9/K14 in the AICDA and PRDM1 promoters in B cells from healthy human individuals (HS) and patients with SLE (ChIP-qPCR analysis). Data are ratios of acetylated H3K9/K14 in the AICDA and PRDM1 promoters of B cells from patients with SLE to that of healthy individuals (set as 1; means ± SD of three healthy individuals and three patients with SLE). ( G ) Intracellular staining of Sirt1 and AID protein levels in spleen B cells from age-matched female C57BL/6 mice, untreated MRL/ Fas lpr/lpr mice, and MRL/ Fas lpr/lpr mice treated with SRT1720 (flow <t>cytometry</t> analysis, one representative of three independent experiments). ( H ) Serum anti-dsDNA and anti-histone IgM, IgG1, and IgG2a titers (ELISAs) in female MRL/ Fas lpr/lpr mice treated with Nil or SRT1720 (means ± SD of six mice in each group). ( I ) Photomicrographs of kidney sections from MRL/ Fas lpr/lpr mice treated with Nil or SRT1720, after hematoxylin and eosin (H E) staining (left) or fluorescence staining of mouse IgG1/IgG2a (right). One representative of three independent experiments yielding similar results. Scale bars, 100 μm. * P
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    Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated promastigotes illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD LSR II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P

    Journal: Frontiers in Microbiology

    Article Title: Leishmanicidal Activity of Piper nigrum Bioactive Fractions is Interceded via Apoptosis In Vitro and Substantiated by Th1 Immunostimulatory Potential In Vivo

    doi: 10.3389/fmicb.2015.01368

    Figure Lengend Snippet: Study of apoptotic correlates post-treatment with bioactive fractions. (A) DNA fragmentation in treated promastigotes illustrated by TUNEL assay. The promastigotes (2 × 10 6 ml -1 ) were treated with bioactive fractions and compounds (500 μg ml -1 ) for 72 h at 22°C after which the cells were processed according to manufacturer’s instructions and acquired on BD LSR II flowcytometer. Histograms depict shift in MFI of (b) PNH, (c) PNE, (d) piperine, and (e) pentamidine with respect to (a) parasite control. (B) Bar graph representing the specific MFI values indicative of TUNEL positivity in PNH, PNE and pentamidine treated samples. ∗∗∗ P

    Article Snippet: The promastigotes were finally washed and resuspended in PBS and acquired on BD LSR-II flow cytometer.

    Techniques: TUNEL Assay

    Flow cytometry of E. coli challenged with antibiotics. At 0 h, 0.5 h, 2 h, and 5 h time points, viability of E. coli MG1655 challenged with ~1× MBC of ampicillin ( A , B ), ~1× MBC of polymyxin B ( C , D ), ~1× MBC of ciprofloxacin ( E , F ), and ~1× MIC of chloramphenicol ( G , H ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F , H ) and a LSR-II flow cytometer ( A , C , E , G ) and by culture-based enumeration ( B , D , F , H ). FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E , G ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F , H ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

    Journal: Microorganisms

    Article Title: Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents

    doi: 10.3390/microorganisms9050924

    Figure Lengend Snippet: Flow cytometry of E. coli challenged with antibiotics. At 0 h, 0.5 h, 2 h, and 5 h time points, viability of E. coli MG1655 challenged with ~1× MBC of ampicillin ( A , B ), ~1× MBC of polymyxin B ( C , D ), ~1× MBC of ciprofloxacin ( E , F ), and ~1× MIC of chloramphenicol ( G , H ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F , H ) and a LSR-II flow cytometer ( A , C , E , G ) and by culture-based enumeration ( B , D , F , H ). FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E , G ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F , H ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

    Article Snippet: We measured antibiotic-treated and untreated samples stained with SYTO 9 and PI at 0.5 h, 2 h, and 5 h exposure times on the Optrode and an LSR-II flow cytometer (BD).

    Techniques: Flow Cytometry, Fluorescence, Staining, Generated, Cell Counting

    Flow cytometry of untreated E. coli and E. coli challenged with isopropanol. At 0 h and 0.25 h time points, viability of E. coli MG1655 challenged with 70% isopropanol ( C , D ) or nothing (untreated) at 37 °C ( A , B ) and 28 °C ( E , F ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F ) and a LSR-II flow cytometer ( A , C , E ) and by culture-based enumeration ( B , D , F ). Measurements of untreated cells at 37 °C were taken at 0 h, 0.5 h, 2 h, and 5 h time points to reflect the time points for antibiotic challenge, while for untreated cells at 28 °C, measurements were taken at 0 h and 0.25 h to reflect the isopropanol challenge. FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

    Journal: Microorganisms

    Article Title: Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents

    doi: 10.3390/microorganisms9050924

    Figure Lengend Snippet: Flow cytometry of untreated E. coli and E. coli challenged with isopropanol. At 0 h and 0.25 h time points, viability of E. coli MG1655 challenged with 70% isopropanol ( C , D ) or nothing (untreated) at 37 °C ( A , B ) and 28 °C ( E , F ) was determined by measuring fluorescence of SYTO 9- and PI-stained cultures using the Optrode ( B , D , F ) and a LSR-II flow cytometer ( A , C , E ) and by culture-based enumeration ( B , D , F ). Measurements of untreated cells at 37 °C were taken at 0 h, 0.5 h, 2 h, and 5 h time points to reflect the time points for antibiotic challenge, while for untreated cells at 28 °C, measurements were taken at 0 h and 0.25 h to reflect the isopropanol challenge. FCM generated a total cell count, live cell count, dying cell count, and dead cell count ( A , C , E ). The dot-dash line represents the limit of quantification for FCM (1 × 10 5 cells/mL). For Optrode measurements, fluorescence intensities were obtained from integrating 505–515 nm for green emissions and 600–610 nm for red emissions. The proportion of live cells in the sample population was determined by the adjusted dye ratio (ADR; B , D , F ). The limit of detection for culture-based enumeration is 500 CFU/mL (dashed line). Data presented is from three biological replicates with a line plotted at the median.

    Article Snippet: We measured antibiotic-treated and untreated samples stained with SYTO 9 and PI at 0.5 h, 2 h, and 5 h exposure times on the Optrode and an LSR-II flow cytometer (BD).

    Techniques: Flow Cytometry, Fluorescence, Staining, Generated, Cell Counting

    Assessment of autophagy. ( A ) Histogram of flow cytometric analysis of mutant cybrids (III-8.48) and control cybrids (C59.32) using CYTOID® Autophagy Detection Kit. Three control and three mutant cybrids were incubated with DMEM in the absence and presence of rapamycin (inducers of autophagy) and chloroquine (lysosomal inhibitor) at 37°C for 18 h, added to CYTO-ID ® -Green dye and analyzed using a Novocyte flow cytometer (ACEA Biosciences). ( B ) Relative ratios of Cyto-ID fluorescence intensity from three mutant and three control cell lines. Three independent determinations were done in each cell line. ( C ) Western blot analysis for autophagic response proteins: LC3-I/II and p62. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β -actin as a loading control. ( D ) Quantification of autophagy markers LC3 I/II and p62 in mutant and control cell lines were determined as described elsewhere ( 70 ). Three independent determinations were done in each cell line. G. Graph details and symbols are explained in the legend to Figure 4 .

    Journal: Nucleic Acids Research

    Article Title: A deafness-associated tRNA mutation caused pleiotropic effects on the m1G37 modification, processing, stability and aminoacylation of tRNAIle and mitochondrial translation

    doi: 10.1093/nar/gkaa1225

    Figure Lengend Snippet: Assessment of autophagy. ( A ) Histogram of flow cytometric analysis of mutant cybrids (III-8.48) and control cybrids (C59.32) using CYTOID® Autophagy Detection Kit. Three control and three mutant cybrids were incubated with DMEM in the absence and presence of rapamycin (inducers of autophagy) and chloroquine (lysosomal inhibitor) at 37°C for 18 h, added to CYTO-ID ® -Green dye and analyzed using a Novocyte flow cytometer (ACEA Biosciences). ( B ) Relative ratios of Cyto-ID fluorescence intensity from three mutant and three control cell lines. Three independent determinations were done in each cell line. ( C ) Western blot analysis for autophagic response proteins: LC3-I/II and p62. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β -actin as a loading control. ( D ) Quantification of autophagy markers LC3 I/II and p62 in mutant and control cell lines were determined as described elsewhere ( 70 ). Three independent determinations were done in each cell line. G. Graph details and symbols are explained in the legend to Figure 4 .

    Article Snippet: Samples were analyzed by BD-LSR II flow cytometer system (Beckton Dickson, Inc.), with an excitation at 488 nm and emission at 529 nm.

    Techniques: Mutagenesis, Incubation, Flow Cytometry, Fluorescence, Western Blot

    Sirt1 dampens the lupus autoantibody response. ( A ) IgG1 + and IgG2a + ANAs in sera from 35-week-old female Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice (immunofluorescence microscopy of one representative of three independent experiments). Scale bar, 50 μm. ( B ) Anti-dsDNA, anti-histone, anti-RNP, and anti-RNA IgG titers (ELISAs) in sera from 35-week-old female Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice (means ± SD of six Aicda cre Sirt1 +/+ and six Aicda cre Sirt1 fl/fl mice, each tested in triplicates). ( C ) Expression of Sirt1 and Aicda (qRT-PCR analysis) in spleen B cells from C57BL/6 mice and lupus-prone MRL/ Fas lpr/lpr and BXD2 mice. Data are relative expression to β -Actin (means ± SD of three mice in each group). Dotted lines depict trends. ( D ) qRT-PCR analysis of expression of SIRT1 and AICDA in B cells from healthy humans and patients with SLE. Data are normalized to expression of β -ACTIN (means ± SD of three healthy individuals and three patients with SLE). Dotted lines depict trends. ( E ) Acetylated H3K9/K14 in the Aicda, Prdm1 , and Xbp1 promoters (ChIP-qPCR analysis) in B cells from C57BL/6 and MRL/ Fas lpr/lpr mice. Data are ratios of acetylated H3K9/K14 in MRL/ Fas lpr/lpr mice to that in C57BL/6 mice (set as 1; means ± SD of three mice in each group). ( F ) Acetylated H3K9/K14 in the AICDA and PRDM1 promoters in B cells from healthy human individuals (HS) and patients with SLE (ChIP-qPCR analysis). Data are ratios of acetylated H3K9/K14 in the AICDA and PRDM1 promoters of B cells from patients with SLE to that of healthy individuals (set as 1; means ± SD of three healthy individuals and three patients with SLE). ( G ) Intracellular staining of Sirt1 and AID protein levels in spleen B cells from age-matched female C57BL/6 mice, untreated MRL/ Fas lpr/lpr mice, and MRL/ Fas lpr/lpr mice treated with SRT1720 (flow cytometry analysis, one representative of three independent experiments). ( H ) Serum anti-dsDNA and anti-histone IgM, IgG1, and IgG2a titers (ELISAs) in female MRL/ Fas lpr/lpr mice treated with Nil or SRT1720 (means ± SD of six mice in each group). ( I ) Photomicrographs of kidney sections from MRL/ Fas lpr/lpr mice treated with Nil or SRT1720, after hematoxylin and eosin (H E) staining (left) or fluorescence staining of mouse IgG1/IgG2a (right). One representative of three independent experiments yielding similar results. Scale bars, 100 μm. * P

    Journal: Science Advances

    Article Title: B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response

    doi: 10.1126/sciadv.aay2793

    Figure Lengend Snippet: Sirt1 dampens the lupus autoantibody response. ( A ) IgG1 + and IgG2a + ANAs in sera from 35-week-old female Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice (immunofluorescence microscopy of one representative of three independent experiments). Scale bar, 50 μm. ( B ) Anti-dsDNA, anti-histone, anti-RNP, and anti-RNA IgG titers (ELISAs) in sera from 35-week-old female Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice (means ± SD of six Aicda cre Sirt1 +/+ and six Aicda cre Sirt1 fl/fl mice, each tested in triplicates). ( C ) Expression of Sirt1 and Aicda (qRT-PCR analysis) in spleen B cells from C57BL/6 mice and lupus-prone MRL/ Fas lpr/lpr and BXD2 mice. Data are relative expression to β -Actin (means ± SD of three mice in each group). Dotted lines depict trends. ( D ) qRT-PCR analysis of expression of SIRT1 and AICDA in B cells from healthy humans and patients with SLE. Data are normalized to expression of β -ACTIN (means ± SD of three healthy individuals and three patients with SLE). Dotted lines depict trends. ( E ) Acetylated H3K9/K14 in the Aicda, Prdm1 , and Xbp1 promoters (ChIP-qPCR analysis) in B cells from C57BL/6 and MRL/ Fas lpr/lpr mice. Data are ratios of acetylated H3K9/K14 in MRL/ Fas lpr/lpr mice to that in C57BL/6 mice (set as 1; means ± SD of three mice in each group). ( F ) Acetylated H3K9/K14 in the AICDA and PRDM1 promoters in B cells from healthy human individuals (HS) and patients with SLE (ChIP-qPCR analysis). Data are ratios of acetylated H3K9/K14 in the AICDA and PRDM1 promoters of B cells from patients with SLE to that of healthy individuals (set as 1; means ± SD of three healthy individuals and three patients with SLE). ( G ) Intracellular staining of Sirt1 and AID protein levels in spleen B cells from age-matched female C57BL/6 mice, untreated MRL/ Fas lpr/lpr mice, and MRL/ Fas lpr/lpr mice treated with SRT1720 (flow cytometry analysis, one representative of three independent experiments). ( H ) Serum anti-dsDNA and anti-histone IgM, IgG1, and IgG2a titers (ELISAs) in female MRL/ Fas lpr/lpr mice treated with Nil or SRT1720 (means ± SD of six mice in each group). ( I ) Photomicrographs of kidney sections from MRL/ Fas lpr/lpr mice treated with Nil or SRT1720, after hematoxylin and eosin (H E) staining (left) or fluorescence staining of mouse IgG1/IgG2a (right). One representative of three independent experiments yielding similar results. Scale bars, 100 μm. * P

    Article Snippet: Flow cytometry of B and T cells, B cell division, surface, and intracellular florescence staining Single-cell suspensions were prepared from mouse spleen and stained with the following antibodies and reagents in different combinations for flow cytometry analysis (LSR-II flow cytometer, BD Biosciences) of B cells (B220+ ), T cells (CD3+ ), germinal center (GL-7hi B220+ ) B cells and plasma cells (B220low CD138+ ), antigen-specific B cells, and class-switched B cells: PE–anti-B220 mAb (clone RA3-6B2; eBioscience), Pacific Blue anti-B220 mAb (clone RA3-6B2; BioLegend), FITC–anti-CD3 mAb (clone 17A2; BioLegend), and biotin–anti-CD138 mAb (clone 281-2; BD Biosciences) followed by FITC-streptavidin (11-4317-87, eBioscience) or PE-streptavidin (12-4317-87, eBioscience), PE-Cy7-anti-CD38 mAB (clone 90; BioLegend), PE– or FITC–anti-IgM mAb (clone RMM-1; BioLegend), FITC–anti-IgG1 mAb (clone A85-1; BD Biosciences), allophycocyanin (APC)–anti-IgG1 mAb (clone X56; BD Biosciences), FITC–anti-IgG3 mAb (clone R40-82; BD Biosciences), FITC–anti-IgA mAb (clone C10-3; BD Biosciences), and biotin–anti-IgD mAb (clone IA6-2; BioLegend) followed by FITC-streptavidin (11-4317-87, eBioscience) or APC-streptavidin (550874, eBioscience), PE-NP4 (N-5070, Biosearch Technologies), and 7-AAD, as all previously described ( , ).

    Techniques: Mouse Assay, Immunofluorescence, Microscopy, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Fluorescence

    Aicda -inducing stimuli down-regulate Sirt1 in human and mouse B cells. ( A ) Sirt1-7 and Aicda expression in mouse naïve B cells before and after stimulation with LPS plus IL-4 for 72 hours, as measured by mRNA-Seq and depicted as RPKM (reads per kilobase of transcripts per million mapped reads; one of two independent experiments yielding comparable results). ( B ) Sirt1 and Aicda transcript levels [quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis] in mouse B cells stimulated with LPS or CD154 plus IL-4 for 0, 6, 12, 24, 48, or 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left panel). Also shown are the inverse correlation scatter plots of Sirt1 and Aicda expression levels (right). ( C ) SIRT1 and AICDA expression (qRT-PCR analysis) in human naïve B cells stimulated with CD154 plus IL-4 and IL-21 for 0, 6, 12, 24, 48, 72, or 96 hours. Data are ratio to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left). Also shown are the inverse correlation scatter plots of SIRT1 and AICDA expression levels (right). ( D ) Sirt1 and Aicda transcript levels (qRT-PCR analysis) in mouse B cells stimulated with anti-Igδ mAb or LPS plus IL-4 for 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( E ) Sirt1 and Aicda expression (qRT-PCR analysis) in spleen B cells from C57BL/6 mice immunized with nil or NP 16 -CGG and analyzed 10 days after immunization. Data are ratios to the expression in nonimmunized mice (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( F ) Sirt1 and AID protein levels in CD19 + IgD hi GL7 − CD138 − naïve B cells, CD19 + IgD − GL7 hi CD138 − germinal center (GC) B cells, and CD19 lo CD138 hi plasma cells/plasmablasts in C57BL/6 mice immunized with NP 16 -CGG, as analyzed by flow cytometry 10 days after immunization. ( G to I ) Sirt1 and AID protein levels in mouse B cells before (nil) and after stimulation with LPS plus IL-4 for 72 hours analyzed by flow cytometry (G), intracellular immunofluorescence (H), and immunoblotting (I). Densitometry quantification of immunoblotting signals normalized to β-actin levels and depicted as ratios of readings in LPS plus IL-4 stimulated B cells to those in unstimulated (0 hours) B cells (I, right). Data in (F) to (I) are one of two independent experiments yielding similar results. *** P

    Journal: Science Advances

    Article Title: B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response

    doi: 10.1126/sciadv.aay2793

    Figure Lengend Snippet: Aicda -inducing stimuli down-regulate Sirt1 in human and mouse B cells. ( A ) Sirt1-7 and Aicda expression in mouse naïve B cells before and after stimulation with LPS plus IL-4 for 72 hours, as measured by mRNA-Seq and depicted as RPKM (reads per kilobase of transcripts per million mapped reads; one of two independent experiments yielding comparable results). ( B ) Sirt1 and Aicda transcript levels [quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis] in mouse B cells stimulated with LPS or CD154 plus IL-4 for 0, 6, 12, 24, 48, or 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left panel). Also shown are the inverse correlation scatter plots of Sirt1 and Aicda expression levels (right). ( C ) SIRT1 and AICDA expression (qRT-PCR analysis) in human naïve B cells stimulated with CD154 plus IL-4 and IL-21 for 0, 6, 12, 24, 48, 72, or 96 hours. Data are ratio to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left). Also shown are the inverse correlation scatter plots of SIRT1 and AICDA expression levels (right). ( D ) Sirt1 and Aicda transcript levels (qRT-PCR analysis) in mouse B cells stimulated with anti-Igδ mAb or LPS plus IL-4 for 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( E ) Sirt1 and Aicda expression (qRT-PCR analysis) in spleen B cells from C57BL/6 mice immunized with nil or NP 16 -CGG and analyzed 10 days after immunization. Data are ratios to the expression in nonimmunized mice (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( F ) Sirt1 and AID protein levels in CD19 + IgD hi GL7 − CD138 − naïve B cells, CD19 + IgD − GL7 hi CD138 − germinal center (GC) B cells, and CD19 lo CD138 hi plasma cells/plasmablasts in C57BL/6 mice immunized with NP 16 -CGG, as analyzed by flow cytometry 10 days after immunization. ( G to I ) Sirt1 and AID protein levels in mouse B cells before (nil) and after stimulation with LPS plus IL-4 for 72 hours analyzed by flow cytometry (G), intracellular immunofluorescence (H), and immunoblotting (I). Densitometry quantification of immunoblotting signals normalized to β-actin levels and depicted as ratios of readings in LPS plus IL-4 stimulated B cells to those in unstimulated (0 hours) B cells (I, right). Data in (F) to (I) are one of two independent experiments yielding similar results. *** P

    Article Snippet: Flow cytometry of B and T cells, B cell division, surface, and intracellular florescence staining Single-cell suspensions were prepared from mouse spleen and stained with the following antibodies and reagents in different combinations for flow cytometry analysis (LSR-II flow cytometer, BD Biosciences) of B cells (B220+ ), T cells (CD3+ ), germinal center (GL-7hi B220+ ) B cells and plasma cells (B220low CD138+ ), antigen-specific B cells, and class-switched B cells: PE–anti-B220 mAb (clone RA3-6B2; eBioscience), Pacific Blue anti-B220 mAb (clone RA3-6B2; BioLegend), FITC–anti-CD3 mAb (clone 17A2; BioLegend), and biotin–anti-CD138 mAb (clone 281-2; BD Biosciences) followed by FITC-streptavidin (11-4317-87, eBioscience) or PE-streptavidin (12-4317-87, eBioscience), PE-Cy7-anti-CD38 mAB (clone 90; BioLegend), PE– or FITC–anti-IgM mAb (clone RMM-1; BioLegend), FITC–anti-IgG1 mAb (clone A85-1; BD Biosciences), allophycocyanin (APC)–anti-IgG1 mAb (clone X56; BD Biosciences), FITC–anti-IgG3 mAb (clone R40-82; BD Biosciences), FITC–anti-IgA mAb (clone C10-3; BD Biosciences), and biotin–anti-IgD mAb (clone IA6-2; BioLegend) followed by FITC-streptavidin (11-4317-87, eBioscience) or APC-streptavidin (550874, eBioscience), PE-NP4 (N-5070, Biosearch Technologies), and 7-AAD, as all previously described ( , ).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay, Flow Cytometry, Immunofluorescence

    Sirt1 overexpression or activation dampens Aicda expression and CSR, while Sirt1 deletion further increases Aicda expression and CSR. ( A ) Ex vivo expression of Sirt1 and Aicda (qRT-PCR) in peripheral blood total B cells isolated from non-intentionally immunized Sirt1 +/+ and Sirt1 super mice. Data are ratios to expression in total B cells from Sirt1 +/+ mice (set as 1; means ± SEM of values from three Sirt1 +/+ mice and three Sirt1 super mice). ( B ) Serum titers [enzyme-linked immunosorbent assay (ELISA)] of total IgM, IgG1, and IgA in non-intentionally immunized Sirt1 +/+ and Sirt1 super mice (means ± SD of six Sirt1 +/+ mice and six Sirt1 super mice). ( C ) Expression of Sirt1, Aicda, Prdm1 , and Xbp1 transcripts (qRT-PCR) in Sirt1 +/+ (set as 1) and Sirt1 super B cells stimulated with LPS plus IL-4 for 72 hours (means ± SEM of three biological independent experiments, each consisting of triplicates; left) as well as immunoblotting and flow cytometry analysis of Sirt1 and AID protein levels in these cells (one of two independent experiments yielding similar results; right). ( D and E ) CSR (D) and plasma cell differentiation (E) in Sirt1 +/+ and Sirt1 super B cells (flow cytometry analysis) after stimulation with appropriate stimuli, as indicated, for 96 hours (left, one representative of three independent experiments; right, means ± SEM of three independent experiments). ( F ) CSR to IgG1 (flow cytometry analysis) in (B220 + GFP + ) B cells transduced by pMIG-GFP or pMIG-GFP-Sirt1 retrovirus (one of two independent experiments yielding similar results). ( G to I ) C57BL/6 B cells stimulated for 96 hours with LPS plus IL-4 in the presence of SRT1720 at indicated doses. Percentages of IgG1 + B cells and B220 low CD138 + plasma cells (G), cell proliferation [carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled cells] (H), and viability [7-aminoactinomycin D–negative (7-AAD − )] (I) (flow cytometry analysis). Data are means ± SEM of three independent experiments (G) or one representative of three independent experiments (H and I). ( J ) Aicda, Prdm1 , and Xbp1 transcript levels (qRT-PCR analysis) in purified B cells treated with nil or SRT1720 at the indicated doses and stimulated for 72 hours with LPS plus IL-4. Data are ratios to the expression in B cells treated with nil (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( K ) Sirt1 transcript levels (qRT-PCR analysis) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 0 and 72 hours. Data are ratios to the expression in unstimulated (0 hours) Aicda cre Sirt1 +/+ B cells (set as 1; ± SEM of three biological independent experiments, each consisting of triplicates). ( L ) CSR to different Ig isotypes (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three independent experiments. ( M ) Expression of indicated genes (qRT-PCR analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 72 hours with LPS plus IL-4 or LPS plus TGF-β and anti-Igδ mAb-dextran (Iα-Cα and Iμ-Cα). Data are ratios to the expression in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( N ) AID protein levels in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 72 hours as analyzed by intracellular staining and flow cytometry. Data are from one of two independent experiments yielding similar results. ( O ) Plasma cell differentiation (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three biological independent experiments. ( P ) Proliferation of spleen B220 + B cells labeled with CFSE and stimulated with LPS plus IL-4 for 72 and 96 hours (top) and B cell viability (7-AAD − , bottom). Data are one representative of three independent experiments yielding similar results. * P

    Journal: Science Advances

    Article Title: B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response

    doi: 10.1126/sciadv.aay2793

    Figure Lengend Snippet: Sirt1 overexpression or activation dampens Aicda expression and CSR, while Sirt1 deletion further increases Aicda expression and CSR. ( A ) Ex vivo expression of Sirt1 and Aicda (qRT-PCR) in peripheral blood total B cells isolated from non-intentionally immunized Sirt1 +/+ and Sirt1 super mice. Data are ratios to expression in total B cells from Sirt1 +/+ mice (set as 1; means ± SEM of values from three Sirt1 +/+ mice and three Sirt1 super mice). ( B ) Serum titers [enzyme-linked immunosorbent assay (ELISA)] of total IgM, IgG1, and IgA in non-intentionally immunized Sirt1 +/+ and Sirt1 super mice (means ± SD of six Sirt1 +/+ mice and six Sirt1 super mice). ( C ) Expression of Sirt1, Aicda, Prdm1 , and Xbp1 transcripts (qRT-PCR) in Sirt1 +/+ (set as 1) and Sirt1 super B cells stimulated with LPS plus IL-4 for 72 hours (means ± SEM of three biological independent experiments, each consisting of triplicates; left) as well as immunoblotting and flow cytometry analysis of Sirt1 and AID protein levels in these cells (one of two independent experiments yielding similar results; right). ( D and E ) CSR (D) and plasma cell differentiation (E) in Sirt1 +/+ and Sirt1 super B cells (flow cytometry analysis) after stimulation with appropriate stimuli, as indicated, for 96 hours (left, one representative of three independent experiments; right, means ± SEM of three independent experiments). ( F ) CSR to IgG1 (flow cytometry analysis) in (B220 + GFP + ) B cells transduced by pMIG-GFP or pMIG-GFP-Sirt1 retrovirus (one of two independent experiments yielding similar results). ( G to I ) C57BL/6 B cells stimulated for 96 hours with LPS plus IL-4 in the presence of SRT1720 at indicated doses. Percentages of IgG1 + B cells and B220 low CD138 + plasma cells (G), cell proliferation [carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled cells] (H), and viability [7-aminoactinomycin D–negative (7-AAD − )] (I) (flow cytometry analysis). Data are means ± SEM of three independent experiments (G) or one representative of three independent experiments (H and I). ( J ) Aicda, Prdm1 , and Xbp1 transcript levels (qRT-PCR analysis) in purified B cells treated with nil or SRT1720 at the indicated doses and stimulated for 72 hours with LPS plus IL-4. Data are ratios to the expression in B cells treated with nil (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( K ) Sirt1 transcript levels (qRT-PCR analysis) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 0 and 72 hours. Data are ratios to the expression in unstimulated (0 hours) Aicda cre Sirt1 +/+ B cells (set as 1; ± SEM of three biological independent experiments, each consisting of triplicates). ( L ) CSR to different Ig isotypes (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three independent experiments. ( M ) Expression of indicated genes (qRT-PCR analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 72 hours with LPS plus IL-4 or LPS plus TGF-β and anti-Igδ mAb-dextran (Iα-Cα and Iμ-Cα). Data are ratios to the expression in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( N ) AID protein levels in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 72 hours as analyzed by intracellular staining and flow cytometry. Data are from one of two independent experiments yielding similar results. ( O ) Plasma cell differentiation (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three biological independent experiments. ( P ) Proliferation of spleen B220 + B cells labeled with CFSE and stimulated with LPS plus IL-4 for 72 and 96 hours (top) and B cell viability (7-AAD − , bottom). Data are one representative of three independent experiments yielding similar results. * P

    Article Snippet: Flow cytometry of B and T cells, B cell division, surface, and intracellular florescence staining Single-cell suspensions were prepared from mouse spleen and stained with the following antibodies and reagents in different combinations for flow cytometry analysis (LSR-II flow cytometer, BD Biosciences) of B cells (B220+ ), T cells (CD3+ ), germinal center (GL-7hi B220+ ) B cells and plasma cells (B220low CD138+ ), antigen-specific B cells, and class-switched B cells: PE–anti-B220 mAb (clone RA3-6B2; eBioscience), Pacific Blue anti-B220 mAb (clone RA3-6B2; BioLegend), FITC–anti-CD3 mAb (clone 17A2; BioLegend), and biotin–anti-CD138 mAb (clone 281-2; BD Biosciences) followed by FITC-streptavidin (11-4317-87, eBioscience) or PE-streptavidin (12-4317-87, eBioscience), PE-Cy7-anti-CD38 mAB (clone 90; BioLegend), PE– or FITC–anti-IgM mAb (clone RMM-1; BioLegend), FITC–anti-IgG1 mAb (clone A85-1; BD Biosciences), allophycocyanin (APC)–anti-IgG1 mAb (clone X56; BD Biosciences), FITC–anti-IgG3 mAb (clone R40-82; BD Biosciences), FITC–anti-IgA mAb (clone C10-3; BD Biosciences), and biotin–anti-IgD mAb (clone IA6-2; BioLegend) followed by FITC-streptavidin (11-4317-87, eBioscience) or APC-streptavidin (550874, eBioscience), PE-NP4 (N-5070, Biosearch Technologies), and 7-AAD, as all previously described ( , ).

    Techniques: Over Expression, Activation Assay, Expressing, Ex Vivo, Quantitative RT-PCR, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Differentiation, Labeling, Purification, Staining

    Activated B cell–specific deletion of residual Sirt1 in Aicda cre Sirt1 fl/fl mice augments the class-switched and hypermutated antibody response. ( A and B ) Serum titers of total IgM, IgG1, and IgA (A) and high-affinity NP 4 -binding IgG1 (B) (ELISA; RU, relative units) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates immunized with NP 16 -CGG at days 0 and 21 at different time points, as indicated ( n = 7 mice in each group). Dotted lines link paired littermates. ( C and D ) AFCs secreting IgM and IgG1 or NP 4 -binding IgG1 (ELISPOTs) in the spleen and bone marrow in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates euthanized 28 days after the first NP 16 -CGG injection. Data are one representative of three independent experiments yielding similar results (C) or means ± SEM of three independent experiments (D). ( E ) Fluorescence microscopy analysis of IgA-producing cells in different gut tissues, as indicated, in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates Aicda cre Sirt1 fl/fl mice (one representative of three independent experiments yielding similar results). Scale bars, 50 μm. ( F ) Overall frequency (change/base) and distribution (pie charts) of point mutations in the V 186.2 region of V 186.2 DJ H -Cγ1 complementary DNA (cDNA; pooled data from two mouse pairs) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice injected (intraperitoneally) with NP 16 -CGG at days 0 and 21 and euthanized at day 28. Also depicted by histograms are frequencies of mutations in the framework (FR) and complementarity-determining (CDR) regions (right). P values were calculated by χ 2 test. ( G and H ) Analysis of class-switched IgM − IgD − IgG + B cells, NP 5 -binding IgG1 + B cells, and NP 5 -binding CD38 + IgG1 + memory B cells (flow cytometry) in spleen (G) and quantification of these cells in the NP 16 -CGG immunized mice (H). ( I ) Flow cytometry analysis of B220 low CD138 + plasmablasts/plasma cells in the spleen and bone marrow. ( J ) Quantification of proportion of B220 low CD138 + plasmablasts/plasma cells among total spleen and bone marrow cells. ( K ) Viable (7-AAD − ) B cells in the spleen (flow cytometry) of the immunized mice. ( L and M ) Flow cytometry analysis of proliferating (incorporating BrdU and BrdU + ) B cells (L) and proportions of T (CD3 + ) cells and B (B220 + ) cells (M). ( N ) Spleen germinal center (B220 + GL7 + CD95 + ) B cells in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice as analyzed by flow cytometry 10 days after NP-CGG injection. ( O ) Quantification of the proportion and of B220 + GL-7 + CD95 + germinal center B cells among total spleen B cells, as analyzed by FACS (left), and total numbers of B220 + GL-7 + CD95 + germinal center B cells in each spleen (right). ( P ) Germinal center structure in the spleen (fluorescence microscopy). Data in (G), (I), (K) to (N), and (P) are one representative of three independent experiments yielding similar results. Scale bar, 100 μm. (H), (J), and (O) are means ± SEM of three or four biological independent experiments. * P

    Journal: Science Advances

    Article Title: B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response

    doi: 10.1126/sciadv.aay2793

    Figure Lengend Snippet: Activated B cell–specific deletion of residual Sirt1 in Aicda cre Sirt1 fl/fl mice augments the class-switched and hypermutated antibody response. ( A and B ) Serum titers of total IgM, IgG1, and IgA (A) and high-affinity NP 4 -binding IgG1 (B) (ELISA; RU, relative units) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates immunized with NP 16 -CGG at days 0 and 21 at different time points, as indicated ( n = 7 mice in each group). Dotted lines link paired littermates. ( C and D ) AFCs secreting IgM and IgG1 or NP 4 -binding IgG1 (ELISPOTs) in the spleen and bone marrow in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates euthanized 28 days after the first NP 16 -CGG injection. Data are one representative of three independent experiments yielding similar results (C) or means ± SEM of three independent experiments (D). ( E ) Fluorescence microscopy analysis of IgA-producing cells in different gut tissues, as indicated, in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mouse littermates Aicda cre Sirt1 fl/fl mice (one representative of three independent experiments yielding similar results). Scale bars, 50 μm. ( F ) Overall frequency (change/base) and distribution (pie charts) of point mutations in the V 186.2 region of V 186.2 DJ H -Cγ1 complementary DNA (cDNA; pooled data from two mouse pairs) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice injected (intraperitoneally) with NP 16 -CGG at days 0 and 21 and euthanized at day 28. Also depicted by histograms are frequencies of mutations in the framework (FR) and complementarity-determining (CDR) regions (right). P values were calculated by χ 2 test. ( G and H ) Analysis of class-switched IgM − IgD − IgG + B cells, NP 5 -binding IgG1 + B cells, and NP 5 -binding CD38 + IgG1 + memory B cells (flow cytometry) in spleen (G) and quantification of these cells in the NP 16 -CGG immunized mice (H). ( I ) Flow cytometry analysis of B220 low CD138 + plasmablasts/plasma cells in the spleen and bone marrow. ( J ) Quantification of proportion of B220 low CD138 + plasmablasts/plasma cells among total spleen and bone marrow cells. ( K ) Viable (7-AAD − ) B cells in the spleen (flow cytometry) of the immunized mice. ( L and M ) Flow cytometry analysis of proliferating (incorporating BrdU and BrdU + ) B cells (L) and proportions of T (CD3 + ) cells and B (B220 + ) cells (M). ( N ) Spleen germinal center (B220 + GL7 + CD95 + ) B cells in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl mice as analyzed by flow cytometry 10 days after NP-CGG injection. ( O ) Quantification of the proportion and of B220 + GL-7 + CD95 + germinal center B cells among total spleen B cells, as analyzed by FACS (left), and total numbers of B220 + GL-7 + CD95 + germinal center B cells in each spleen (right). ( P ) Germinal center structure in the spleen (fluorescence microscopy). Data in (G), (I), (K) to (N), and (P) are one representative of three independent experiments yielding similar results. Scale bar, 100 μm. (H), (J), and (O) are means ± SEM of three or four biological independent experiments. * P

    Article Snippet: Flow cytometry of B and T cells, B cell division, surface, and intracellular florescence staining Single-cell suspensions were prepared from mouse spleen and stained with the following antibodies and reagents in different combinations for flow cytometry analysis (LSR-II flow cytometer, BD Biosciences) of B cells (B220+ ), T cells (CD3+ ), germinal center (GL-7hi B220+ ) B cells and plasma cells (B220low CD138+ ), antigen-specific B cells, and class-switched B cells: PE–anti-B220 mAb (clone RA3-6B2; eBioscience), Pacific Blue anti-B220 mAb (clone RA3-6B2; BioLegend), FITC–anti-CD3 mAb (clone 17A2; BioLegend), and biotin–anti-CD138 mAb (clone 281-2; BD Biosciences) followed by FITC-streptavidin (11-4317-87, eBioscience) or PE-streptavidin (12-4317-87, eBioscience), PE-Cy7-anti-CD38 mAB (clone 90; BioLegend), PE– or FITC–anti-IgM mAb (clone RMM-1; BioLegend), FITC–anti-IgG1 mAb (clone A85-1; BD Biosciences), allophycocyanin (APC)–anti-IgG1 mAb (clone X56; BD Biosciences), FITC–anti-IgG3 mAb (clone R40-82; BD Biosciences), FITC–anti-IgA mAb (clone C10-3; BD Biosciences), and biotin–anti-IgD mAb (clone IA6-2; BioLegend) followed by FITC-streptavidin (11-4317-87, eBioscience) or APC-streptavidin (550874, eBioscience), PE-NP4 (N-5070, Biosearch Technologies), and 7-AAD, as all previously described ( , ).

    Techniques: Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Injection, Fluorescence, Microscopy, Flow Cytometry, FACS

    Increased glucose concentration reduces cytosolic NAD + , increases acetylation of Aicda promoter histones, Dnmt1 and NF-κB p65, and enhances Aicda expression and CSR. B cells were cultured in glucose-free fetal bovine serum (FBS)–RPMI medium supplemented with increased concentrations of ( A ) glucose or ( B ) galactose or in complete FBS-RPMI-1640 medium supplemented with increased concentrations of ( C ) 2-DG or ( D ) NAD + and stimulated with LPS plus IL-4. Surface expression of B220 and IgG1 was analyzed by flow cytometry after 96 hours of culture. Expression of Aicda, Prdm1 , and Irf4 was analyzed by qRT-PCR after 72 hours of culture. NAD + and NADH concentrations in B cells cultured with increased amount of glucose were also determined after 72 hours. Data are from one representative of three independent experiments yielding comparable results (left) or means ± SEM of three biological independent experiments, each consisting of triplicates (right). C57BL/6 ( E to L ) or Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl ( M and N ) B cells were cultured in glucose-free FBS-RPMI medium supplied with indicated concentrations of glucose and stimulated with LPS plus IL-4 for 72 (E to M) or 96 hours (N). (E and F) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1 , and Xbp1 promoters (E) and recruitment of Sirt1 to the Aicda promoter (F) in B cells cultured with 0 or 20 mM of glucose. Data are ratios to recruitment in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (G) Densitometry quantification of immunoblotting signals of acetylated Dnmt1 and total Dnmt1 after normalization to β-actin levels in B cells. Data are ratios of acetylated Dnmt1 and Dnmt1 in B cells cultured with increased concentrations of glucose to B cells cultured without glucose (set as 1; means ± SD of three independent experiments). (H) ChIP-qPCR analysis of recruitment of Dnmt1 to the Aicda promoter in B cells cultured in different concentrations of glucose, as indicated. Data are ratios of recruitment in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (I) MeDIP-qPCR analysis of DNA methylation in the Aicda promoter of B cells cultured in different concentrations of glucose, as indicated. Data are ratios of values in B cells cultured in glucose to those in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (J and K) Immunoblotting analysis of acetylated NF-κB p65 and total NF-κB p65 in B cells cultured in different concentrations of glucose, as indicated (J); densitometry quantification of signals expressed as ratios of signal in B cells cultured in glucose to that in B cells cultured without glucose (set as 1; means ± SD of three independent experiments) (K). (L) Recruitment of acetylated NF-κB p65 to the Aicda promoter (ChIP-qPCR analysis) in induced B cells. Data are ratios of recruitment of acetylated NF-κB p65 to Aicda promoter in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments). (M) qRT-PCR analysis of Aicda expression in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in the different concentrations of glucose. Data are ratios of Aicda expression in Aicda cre Sirt1 fl/fl B cells to that in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (N) Flow cytometry analysis of CSR to IgG1 in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in different concentrations of glucose. Data are ratios of IgG1 + Aicda cre Sirt1 fl/fl B cells to Aicda cre Sirt1 +/+ B cells (set as 1 in each glucose concentration; means ± SEM of three biological independent experiments). * P

    Journal: Science Advances

    Article Title: B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response

    doi: 10.1126/sciadv.aay2793

    Figure Lengend Snippet: Increased glucose concentration reduces cytosolic NAD + , increases acetylation of Aicda promoter histones, Dnmt1 and NF-κB p65, and enhances Aicda expression and CSR. B cells were cultured in glucose-free fetal bovine serum (FBS)–RPMI medium supplemented with increased concentrations of ( A ) glucose or ( B ) galactose or in complete FBS-RPMI-1640 medium supplemented with increased concentrations of ( C ) 2-DG or ( D ) NAD + and stimulated with LPS plus IL-4. Surface expression of B220 and IgG1 was analyzed by flow cytometry after 96 hours of culture. Expression of Aicda, Prdm1 , and Irf4 was analyzed by qRT-PCR after 72 hours of culture. NAD + and NADH concentrations in B cells cultured with increased amount of glucose were also determined after 72 hours. Data are from one representative of three independent experiments yielding comparable results (left) or means ± SEM of three biological independent experiments, each consisting of triplicates (right). C57BL/6 ( E to L ) or Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl ( M and N ) B cells were cultured in glucose-free FBS-RPMI medium supplied with indicated concentrations of glucose and stimulated with LPS plus IL-4 for 72 (E to M) or 96 hours (N). (E and F) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1 , and Xbp1 promoters (E) and recruitment of Sirt1 to the Aicda promoter (F) in B cells cultured with 0 or 20 mM of glucose. Data are ratios to recruitment in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (G) Densitometry quantification of immunoblotting signals of acetylated Dnmt1 and total Dnmt1 after normalization to β-actin levels in B cells. Data are ratios of acetylated Dnmt1 and Dnmt1 in B cells cultured with increased concentrations of glucose to B cells cultured without glucose (set as 1; means ± SD of three independent experiments). (H) ChIP-qPCR analysis of recruitment of Dnmt1 to the Aicda promoter in B cells cultured in different concentrations of glucose, as indicated. Data are ratios of recruitment in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (I) MeDIP-qPCR analysis of DNA methylation in the Aicda promoter of B cells cultured in different concentrations of glucose, as indicated. Data are ratios of values in B cells cultured in glucose to those in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (J and K) Immunoblotting analysis of acetylated NF-κB p65 and total NF-κB p65 in B cells cultured in different concentrations of glucose, as indicated (J); densitometry quantification of signals expressed as ratios of signal in B cells cultured in glucose to that in B cells cultured without glucose (set as 1; means ± SD of three independent experiments) (K). (L) Recruitment of acetylated NF-κB p65 to the Aicda promoter (ChIP-qPCR analysis) in induced B cells. Data are ratios of recruitment of acetylated NF-κB p65 to Aicda promoter in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments). (M) qRT-PCR analysis of Aicda expression in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in the different concentrations of glucose. Data are ratios of Aicda expression in Aicda cre Sirt1 fl/fl B cells to that in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (N) Flow cytometry analysis of CSR to IgG1 in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in different concentrations of glucose. Data are ratios of IgG1 + Aicda cre Sirt1 fl/fl B cells to Aicda cre Sirt1 +/+ B cells (set as 1 in each glucose concentration; means ± SEM of three biological independent experiments). * P

    Article Snippet: Flow cytometry of B and T cells, B cell division, surface, and intracellular florescence staining Single-cell suspensions were prepared from mouse spleen and stained with the following antibodies and reagents in different combinations for flow cytometry analysis (LSR-II flow cytometer, BD Biosciences) of B cells (B220+ ), T cells (CD3+ ), germinal center (GL-7hi B220+ ) B cells and plasma cells (B220low CD138+ ), antigen-specific B cells, and class-switched B cells: PE–anti-B220 mAb (clone RA3-6B2; eBioscience), Pacific Blue anti-B220 mAb (clone RA3-6B2; BioLegend), FITC–anti-CD3 mAb (clone 17A2; BioLegend), and biotin–anti-CD138 mAb (clone 281-2; BD Biosciences) followed by FITC-streptavidin (11-4317-87, eBioscience) or PE-streptavidin (12-4317-87, eBioscience), PE-Cy7-anti-CD38 mAB (clone 90; BioLegend), PE– or FITC–anti-IgM mAb (clone RMM-1; BioLegend), FITC–anti-IgG1 mAb (clone A85-1; BD Biosciences), allophycocyanin (APC)–anti-IgG1 mAb (clone X56; BD Biosciences), FITC–anti-IgG3 mAb (clone R40-82; BD Biosciences), FITC–anti-IgA mAb (clone C10-3; BD Biosciences), and biotin–anti-IgD mAb (clone IA6-2; BioLegend) followed by FITC-streptavidin (11-4317-87, eBioscience) or APC-streptavidin (550874, eBioscience), PE-NP4 (N-5070, Biosearch Technologies), and 7-AAD, as all previously described ( , ).

    Techniques: Concentration Assay, Expressing, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylated DNA Immunoprecipitation, DNA Methylation Assay