Journal: Frontiers in Immunology

Article Title: Sestrin2 Suppresses Classically Activated Macrophages-Mediated Inflammatory Response in Myocardial Infarction through Inhibition of mTORC1 Signaling

doi: 10.3389/fimmu.2017.00728

Figure Lengend Snippet: Ectopic Sestrin2 expression in M1 macrophages attenuates post-MI inflammation and favors tissue repair in hearts. (A) Macrophage abundance in infarcted myocardium at day 3 after myocardial infarction (MI) (day 4 after Clophosome ® treatment). Ctrl: un-pretreated mice. Depletion: Clophosome ® injection. Depletion + M (L-C): Clophosome ® injection followed by transfer of M1 macrophages transduced with control lentivirus. Depletion + M (L-S): Clophosome ® injection followed by transfer of M1 macrophages transduced with SESN2 lentiviral activation particles. Numbers in the plots are proportions of gated cells populations. This is a representative of two independent experiments. (B,C) mRNA abundance of indicated cytokines and chemokines in infarcted myocardium. (D,E) Percentage of Gr-1 + neutrophils in the whole cardiac cells at day 3 after MI. Representative flow cytometry dot plots are shown in (D) , and statistics is shown in (E) . (F) Masson staining at day 7 after MI. Left panel: representative images. Right panel: statistics of collagen volume fraction (CVF). N = 4–5 per group. * p

Article Snippet: Flow Cytometry Analysis Anti-mouse antibodies were used: APC anti-CD3 (17A2), APC anti-B220 (RA3-6B2), APC anti-Gr1 (RB6-8C5), APC anti-CD90 (OX-7), FITC anti-CD11b (M1/70), PE anti-F4/80 (T45-2342), and PE-Cy7 anti-Ly-6C (AL-21) were purchased from BD Biosciences.

Techniques: Expressing, Mouse Assay, Injection, Transduction, Activation Assay, Flow Cytometry, Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Sestrin2 Suppresses Classically Activated Macrophages-Mediated Inflammatory Response in Myocardial Infarction through Inhibition of mTORC1 Signaling

doi: 10.3389/fimmu.2017.00728

Figure Lengend Snippet: Ectopic Sestrin2 expression in M1 macrophages inhibits pro-inflammatory response in vitro . (A) Expression of CD80 and CD163 on in vitro cultured M1 and M2 monocyte-derived macrophages. Numbers in the quadrants are proportions of corresponding subpopulations. This is a representative of two independent experiments. (B) Lentiviral transduction efficiency of cultured macrophages is detected with flow cytometry. Note that here macrophages were not transduced with GFP-free SESN2 lentiviral activation particles. Instead, they were transduced by copGFP lentiviral particles containing GFP sequence. Numbers in the histogram are percentages of GFP + cells. (C) Sestrin2 protein levels in M1 and M2 macrophages after lentiviral transduction. L-C: control lentivirus not inducing Sestrin2 expression. L-S: SESN2 lentiviral activation particles. This is a representative of three independent experiments. (D–F) mRNA abundance of TNF-α, IL-1β, and IL-10 in lentivirus-transduced M1 and M2 macrophages with or without lipopolysaccharide (LPS) stimulation. M1 L-C: M1 macrophages transduced with control lentivirus. M1 L-S: M1 macrophages transduced with SESN2 lentiviral activation particles. M2 L-C: M2 macrophages transduced with control lentivirus. M2 L-S: M2 macrophages transduced with SESN2 lentiviral activation particles. N = 6 per group. * p

Article Snippet: Flow Cytometry Analysis Anti-mouse antibodies were used: APC anti-CD3 (17A2), APC anti-B220 (RA3-6B2), APC anti-Gr1 (RB6-8C5), APC anti-CD90 (OX-7), FITC anti-CD11b (M1/70), PE anti-F4/80 (T45-2342), and PE-Cy7 anti-Ly-6C (AL-21) were purchased from BD Biosciences.

Techniques: Expressing, In Vitro, Cell Culture, Derivative Assay, Transduction, Flow Cytometry, Cytometry, Activation Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: Sestrin2 Suppresses Classically Activated Macrophages-Mediated Inflammatory Response in Myocardial Infarction through Inhibition of mTORC1 Signaling

doi: 10.3389/fimmu.2017.00728

Figure Lengend Snippet: Sestrin2 inhibits mTORC1 signal pathway in M1 macrophages. (A) mTOR phosphorylation in transferred M1 macrophages. M1 macrophages were transduced with lentivirus and transferred into recipient mice as in Figure 5 . These macrophages were then sorted using flow cytometry from infarcted myocardium at day 3 after myocardial infarction (MI) for detection of mTORC1 activation status. L-C: M1 macrophages transduced with control lentivirus. L-S: M1 macrophages transduced with SESN2 lentiviral activation particles. #1 to #3 indicate three mice. (B) mTOR phosphorylation in in vitro cultured M1 macrophages after lentiviral transduction. (C) 4EBP1 phosphorylation in transferred M1 macrophages at day 3 after MI. N = 3 per group. (D) mTOR phosphorylation in in vitro cultured M1 macrophages in the presence or absence of 1-h MHY1485 treatment. L-C: M1 macrophages transduced with control lentivirus. L-S: M1 macrophages transduced with SESN2 lentiviral activation particles. This is a representative image of two independent experiments. (E–G) mRNA abundance of indicated cytokines in in vitro cultured M1 macrophages. LPS: LPS stimulation. M + LPS: MHY1485 pretreatment followed by LPS stimulation. N = 5 per group. * p

Article Snippet: Flow Cytometry Analysis Anti-mouse antibodies were used: APC anti-CD3 (17A2), APC anti-B220 (RA3-6B2), APC anti-Gr1 (RB6-8C5), APC anti-CD90 (OX-7), FITC anti-CD11b (M1/70), PE anti-F4/80 (T45-2342), and PE-Cy7 anti-Ly-6C (AL-21) were purchased from BD Biosciences.

Techniques: Transduction, Mouse Assay, Flow Cytometry, Cytometry, Activation Assay, In Vitro, Cell Culture

Journal: Frontiers in Immunology

Article Title: Sestrin2 Suppresses Classically Activated Macrophages-Mediated Inflammatory Response in Myocardial Infarction through Inhibition of mTORC1 Signaling

doi: 10.3389/fimmu.2017.00728

Figure Lengend Snippet: Sestrin2 is expressed in cardiac macrophages after myocardial infarction (MI). (A) Representative flow cytometry dot plots showing cardiac macrophage subpopulations in infarcted myocardium at day 1, day 3, day 5, and day 7 after MI. Numbers in the quadrants are proportions of corresponding subpopulations. Sham: Sham-operated animal. This is a representative of three independent experiments. (B) Sestrin2 protein levels in total cardiac macrophages (upper panel) and blood monocytes (lower panel). (C) Statistics for (B) . N = 3 per group. * p

Article Snippet: Flow Cytometry Analysis Anti-mouse antibodies were used: APC anti-CD3 (17A2), APC anti-B220 (RA3-6B2), APC anti-Gr1 (RB6-8C5), APC anti-CD90 (OX-7), FITC anti-CD11b (M1/70), PE anti-F4/80 (T45-2342), and PE-Cy7 anti-Ly-6C (AL-21) were purchased from BD Biosciences.

Techniques: Flow Cytometry, Cytometry

Journal: Oncoimmunology

Article Title: The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

doi: 10.1080/2162402X.2016.1245267

Figure Lengend Snippet: miR-26a reduces CTL cytotoxicity against tumor cells in vitro . (A) Relative gene expression values for miR-26a in CTLs from Mir26a -Tg mice. CTLs from WT mice were used as a control (n = 5). (B) MACS-purified naïve CD8 + T cells were activated using anti-CD3 and anti-CD28 antibodies for 2 d, and flow cytometry was used to assess CTL effector molecule expression. Both the expression percentage and MFI are shown (n = 5). (C, D) qPCR analysis of granzyme B and IFNγ expression was also performed in CTLs from Mir26a -Tg mice and WT mice (n = 5). (E) CTLs from WT or Mir26a -Tg mice were incubated with target cells (EL4 cells) for 6 h. The ratios of target cells: effector cells were 1:1, 1:5, 1:10, and 1:20. Error bars in the curve represent the SD among four mice. Statistically-significant differences were measured using an unpaired Student's t -test (A, C, D, and E) and a paired Student's t -test (B), * p

Article Snippet: After 4 h, cell suspensions were stained using the Live/Dead Violet Viability Kit (Invitrogen, Carlsbad, CA) to remove dead cells and surface stained for CD8+ , Thy1.1 or Thy1.2 using anti-mouse flow cytometry antibodies (Biolegend, CA, USA).

Techniques: CTL Assay, In Vitro, Expressing, Mouse Assay, Magnetic Cell Separation, Purification, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Incubation

Journal: Oncoimmunology

Article Title: The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

doi: 10.1080/2162402X.2016.1245267

Figure Lengend Snippet: miR-26a Decoy is sufficient to rescue TME-suppressed CTL function in vitro. (A, B) Naïve pMel-1 CTLs were activated using 5 μM gp100 25–33 peptide for 2 d, followed by retroviral transduction with a mock decoy or the miR-26a decoy vector and treated with or without lyophilized CM from B16 melanoma cells for an additional 48 h. Flow cytometry analysis was used for CTL effector molecule expression in vitro (n = 5). (C) In vitro cytotoxicity of mock or miR-26a decoy CTLs. CTLs were cultured with target cells at a 10:1 ratio (n = 5). Statistically -significant differences were measured using an unpaired Student's t -test, * p

Article Snippet: After 4 h, cell suspensions were stained using the Live/Dead Violet Viability Kit (Invitrogen, Carlsbad, CA) to remove dead cells and surface stained for CD8+ , Thy1.1 or Thy1.2 using anti-mouse flow cytometry antibodies (Biolegend, CA, USA).

Techniques: CTL Assay, In Vitro, Transduction, Plasmid Preparation, Flow Cytometry, Cytometry, Expressing, Cell Culture

Journal: Oncoimmunology

Article Title: The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

doi: 10.1080/2162402X.2016.1245267

Figure Lengend Snippet: miR-26a Decoy improves the efficacy of CTL adoptive transfer therapy. (A) Six days after subcutaneous injection of 2 × 10 5 B16/F10 cells into the right lateral flank of C57BL/6 recipient mice, tumor-bearing mice received adoptive transfer of 2 × 10 5 mock or miR-26a decoy Thy1.1 + PMel-1 CTLs. Mice were monitored regularly and tumor volumes were calculated. n=12 per group. (B, C) Tumor cell suspensions were prepared by digestion with the Papain dissociation system. Flow cytometry analysis was performed to assess the effector responses of Thy1.1 + pMel-1 CTLs. Both expression frequency (B) and MFI (C) were calculated (n = 7). (D, E) Tumor-bearing mice received an adoptive transfer of 2 × 10 5 miR-26a decoy CTLs with GSK343 treatment for 2 d. Flow cytometry analysis was performed to assess the effector response of Thy1.1 + pMel-1 CTLs. Both expression percentage and MFI were calculated (n = 6). Statistically significant differences were determined using an unpaired Student's t -test, * p

Article Snippet: After 4 h, cell suspensions were stained using the Live/Dead Violet Viability Kit (Invitrogen, Carlsbad, CA) to remove dead cells and surface stained for CD8+ , Thy1.1 or Thy1.2 using anti-mouse flow cytometry antibodies (Biolegend, CA, USA).

Techniques: CTL Assay, Adoptive Transfer Assay, Injection, Mouse Assay, Flow Cytometry, Cytometry, Expressing

Journal: Scientific Reports

Article Title: Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury

doi: 10.1038/srep36365

Figure Lengend Snippet: NKT cells in SPF and GF mice have similar function. ( a , b ) αGalCer (250 μg/kg) or vehicle were injected into BALB/c mice, samples were analyzed 24 hr post-injection. ( a ) Serum ALT and AST levels. ( b ) Liver IFN-γ and IL-4 levels were measured by ELISA. ( c , d ) Splenic cells from untreated SPF and GF mice were cultured with the stimulation of either αGalCer or vehicle for 24 hr. ( c ) IFN-γ and IL-4 levels in the supernatant were analyzed by Elisa. ( d ) Intracellular IFN-γ expression in NKT cells were analyzed by flow cytometry. The data represent means ± SEM (n = 8), ns represents not significant, One-way ANOVA.

Article Snippet: Flow Cytometry Anti-mouse αGalCer:CD1d complex antibody (L363, eBioscience, San Diego, CA) was used to detect glycolipid antigen presentation on CD1d.

Techniques: Mouse Assay, Injection, AST Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Flow Cytometry, Cytometry

Journal: Scientific Reports

Article Title: Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury

doi: 10.1038/srep36365

Figure Lengend Snippet: ConA is unable to activate NKT cells from GF mice. BALB/c mice were sacrificed 24 hr after ConA or PBS injection. ( a – c ) Intrahepatic leukocyte was analyzed by flow cytometry. ( a ) The percentages of NKT cells (CD19 − CD3 + αGalCer/CD1d tetramer + ) were determined, the representative scatterplot (left panel) and summative histogram (right panel) are shown. ( b ) Measurements of CD69 expression on hepatic NKT cells. ( c ) The intracellular IFN-γ levels in hepatic NKT cells, the filled flow cytometric histograms represent PBS-treated mice, and the open histograms represent ConA-treated mice. ( d ) OPN levels in the liver tissue, analyzed by Elisa. Two independent experiments with similar results were performed. The data represent means ± SEM (n = 8, two independent experiments). **P

Article Snippet: Flow Cytometry Anti-mouse αGalCer:CD1d complex antibody (L363, eBioscience, San Diego, CA) was used to detect glycolipid antigen presentation on CD1d.

Techniques: Mouse Assay, Injection, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury

doi: 10.1038/srep36365

Figure Lengend Snippet: Levels of CD1d-presented and un-presented glycolipids are substantially lower in GF mice. ( a , b ) SPF and GF BALB/c mice were sacrificed 24 hr after ConA or PBS injection. ( a ) Using mAb L363, we detected the percentage of glycolipid/CD1d complexes in the intrahepatic leukocytes, gated on total leukocytes. ( b ) The mean fluorescence intensity (MFI) of glycolipid/CD1d complexes on total intrahepatic leukocytes and DCs. ( c ) To compared the glycolipids presenting ability between SPF and GF mice, splenic cells from untreated SPF and GF BALB/c mice were cultured with either αGalCer or vehicle stimulation for 4 and 24 hr. Then used L363 to analysis the glycolipid/CD1d complex expression on DCs by flow cytometry, filled flow cytometric histograms represent cultured with vehicle, and opened histograms represent cultured with αGalCer. ( d ) Mice were sacrificed 24 hr post αGalCer (250 μg/kg) or vehicle injection. Using L363, the glycolipid/CD1d complex on hepatic DCs was analyzed by flow cytometry. ( e ) Plasma LPS levels of SPF and GF mice 24 hr after ConA or PBS injection. The data represent means ± SEM (n = 8, two independent experiments), **P

Article Snippet: Flow Cytometry Anti-mouse αGalCer:CD1d complex antibody (L363, eBioscience, San Diego, CA) was used to detect glycolipid antigen presentation on CD1d.

Techniques: Mouse Assay, Injection, Fluorescence, Cell Culture, Expressing, Flow Cytometry, Cytometry

Journal: Scientific Reports

Article Title: Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury

doi: 10.1038/srep36365

Figure Lengend Snippet: Analyze of APCs phenotypes and CD1d expression. Liver infiltrating leukocytes were prepared from SPF and GF BALB/c mice 24 hr after ConA or PBS injection. The percentages of CD11c + DCs after ( a ) PBS or ( b ) ConA treatment were determined by flow cytometry. The CD1d expression of ( c ) total intrahepatic leukocytes, ( d ) DCs (CD11c + ) and ( e ) macrophage cells. The data represent means ± SEM (n = 8, two independent experiments), ns represents not significant, ( a , b ) Mann-Whitney U test, ( c–e ) One-way ANOVA.

Article Snippet: Flow Cytometry Anti-mouse αGalCer:CD1d complex antibody (L363, eBioscience, San Diego, CA) was used to detect glycolipid antigen presentation on CD1d.

Techniques: Expressing, Mouse Assay, Injection, Flow Cytometry, Cytometry, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury

doi: 10.1038/srep36365

Figure Lengend Snippet: ConA-induced liver injury is restored in bacteria-treated GF mice. Except for the ‘ConA+PBS’ group, other groups were pretreated with a mixture of killed intestinal bacteria (Int Bact) prior to ConA injection by intragastric gavage (ig) or intravenous injection (iv), 8 GF BALB/c mice per group. “ConA + PBS” group received 8 day PBS orally administration followed by ConA injection, “PBS + Int Bact ig” group received 8 day Int Bact orally administration followed by PBS injection. “ConA + Int Bact ig” group received 8 day Int Bact orally administration followed by ConA injection. “ConA + Int Bact iv” group received an Int Bact injection followed by ConA injection (see details in the supplementary methods ). Int Bact was consisted of E. coli , E. faecalis , Lactobacillus , Salmonella enteritidis and Group A Streptococcus . ( a ) H E staining of livers. Original magnification, 200×, scale bars represents 50 μm. ( b ) Serum ALT and AST levels. ( c ) Percentages of hepatic NKT cells in intrahepatic leukocytes. ( d ) Intracellular IFN-γ levels in hepatic NKT cells, filled flow cytometric histograms represent GF mice with PBS treatment before ConA injection, and open histograms represent GF mice pretreated with Int Bact before ConA injection. ( e ) The percentages of IFN-γ + NKT cells in total hepatic NKT cells. ( f ) The level of glycolipid/CD1d complex of total intrahepatic leukocytes, detected by L363 through flow cytometry. The data represent means ± SEM (n = 8, two independent experiments), *P

Article Snippet: Flow Cytometry Anti-mouse αGalCer:CD1d complex antibody (L363, eBioscience, San Diego, CA) was used to detect glycolipid antigen presentation on CD1d.

Techniques: Mouse Assay, Injection, Staining, AST Assay, Flow Cytometry, Cytometry

Journal: Scientific Reports

Article Title: Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury

doi: 10.1038/srep36365

Figure Lengend Snippet: Intestinal bacteria contain NKT recognized glycolipids. ( a , b ) Sorted liver DCs from untreated SPF BALB/c mice were stimulated for 24 hr with αGalCer (200 ng), Int Bact or vehicle, respectively. Using L363, the presented glycolipids on CD1d were analyzed by flow cytometry, ( a ) the flow cytometric figure and ( b ) the statistical result were showed. ( c ) Staining of liver-infiltrating leukocytes from wild-type SPF C57BL/6 mice (WT) or CD1d-deficient mice with antigen-loaded CD1d dimer that constructed by loading CD1d:Ig protein with αGalCer, Int Bact, LPS or vehicle, respectively (see detail in methods). The data represent means ± SEM (n = 8, two independent experiments), ***P

Article Snippet: Flow Cytometry Anti-mouse αGalCer:CD1d complex antibody (L363, eBioscience, San Diego, CA) was used to detect glycolipid antigen presentation on CD1d.

Techniques: Mouse Assay, Flow Cytometry, Cytometry, Staining, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CD31 signals confer immune privilege to the vascular endothelium

doi: 10.1073/pnas.1509627112

Figure Lengend Snippet: CD31 does not affect the expression of PDL-1 or Spi6 or antioxidant activity by ECs. ( A ) Surface expression of the inhibitory coreceptor PDL-1 by untreated or TNF-α–treated WT and CD31-KO ECs was assessed by flow cytometry. ( B ) Expression

Article Snippet: For flow cytometry staining rat anti-mouse CD31 antibody clone 390 (eBioscience) was used.

Techniques: Expressing, Antioxidant Activity Assay, Flow Cytometry, Cytometry

Journal: Journal of Virology

Article Title: HIV Protease-Generated Casp8p41, When Bound and Inactivated by Bcl2, Is Degraded by the Proteasome

doi: 10.1128/JVI.00037-18

Figure Lengend Snippet: Proteasome inhibitors increase Casp8p41 levels and kill HIV-infected CD4 T cell cultures more than uninfected cultures. (A) Uninfected primary CD4 + T cells were treated with bortezomib or ixazomib at increasing concentrations for 48 h, and cell death was assessed by activated caspase 3 detection by intracellular flow cytometry. Depicted are the means and SD of the results of two experiments. (B and C) Jurkat CD4 + T cells were transfected with empty vector or GFP-Casp8p41 and then treated with control (DMSO), bortezomib, or ixazomib, and the percentage of cells that were GFP positive was analyzed 6 h later. (C) Mean (plus SD) data from three independent replicates of the experiment shown in panel B compared by a Kruskal-Wallis test. (D) Primary CD4 T cells were infected with HIV IIIb , treated with control or bortezomib, and assessed for intracellular Casp8p41 expression by flow cytometry. (E) Primary CD4 T cells were infected with HIV-Luc; treated with DMSO, bortezomib, or ixazomib; and monitored for viability by ATP content over time. (F) Primary CD4 T cells infected with HIV-Luc were treated with DMSO, bortezomib, or ixazomib and monitored for luciferase activity over time. (G and H) Jurkat T cells were mock or GFP-HIV infected, treated with DMSO or ixazomib, and monitored for GFP (G) or cell death (by cell permeability dye) (H) over time. (I and J) Cell death selectively measured in GFP-expressing cells (I) and GFP-negative cells (J) in panels G and H was monitored over time. *, P

Article Snippet: The following antibodies were used for flow cytometry: phycoerythrin (PE)-conjugated mouse anti-HIV core antibody (Beckman Coulter) and allophycocyanin (APC)-conjugated rabbit anti-active caspase 3 antibody (BD Pharmingen; 1:100).

Techniques: Infection, Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Permeability

Journal: PLoS ONE

Article Title: Human Adipose-Tissue Derived Stromal Cells in Combination with Hypoxia Effectively Support Ex Vivo Expansion of Cord Blood Haematopoietic Progenitors

doi: 10.1371/journal.pone.0124939

Figure Lengend Snippet: Characteristics of cbHSPCs-1 population at 20% O 2 and 5% O 2 . Floating cbHSPCs-1 were harvested on Day 7. CD45, CD34 and CD133 positive cells were evaluated with flow cytometry and the number of CFCs was estimated in MetoCult H4534. A. Representative histograms of cbHSPCs immunostaining. Isotypic control—black line, grey fill, positively stained cells—black line. B. Enrichment of cbHSPC-1 population with low differentiated hematopoietic precursors. The data are presented as M±S.E.M (n = 4). *—p

Article Snippet: Flow Cytometry Mouse anti-human monoclonal antibodies for CD45, CD34, CD73, CD90, CD105, (BD Biosciences, USA), CD133/1 (AC133) (Miltenyi-Biotec, Germany) and isotype IgGs, conjugated with FITC or PE were used for ASCs and cbHSPCs immunophenotyping.

Techniques: Flow Cytometry, Cytometry, Immunostaining, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation

doi: 10.4049/jimmunol.1302874

Figure Lengend Snippet: CD151 deficiency does not alter basal phenotype of cultured BMMCs. ( A ) CD151 deficiency does not affect growth dynamics of mast cell culture. WT and CD151 −/− BMMCs were cultured in IL-3–conditioned media for 8 wk and the total numbers of live cells in culture were counted at each time point indicated ( left ). Flow cytometry for CD151 expression on peritoneal mast cells (flow cytometry chart) ( middle ), qPCR detection of CD151 mRNA in WT and CD151 −/− BMMCs (bar graph, right ), and Western blot analysis of total-protein lysates for CD151 expression (Western blot, right ) all confirm constitutive CD151 expression in WT mast cells. WT peritoneal mast cells are shown as gray-filled histogram with dotted line and negative control as transparent histogram with dotted line. In immunoblotting, actin was used as a loading control. ( B ) Five-week-old BMMCs from WT and CD151 −/− mice were stained with toluidine blue and images were obtained with an original magnification of ×100. Flow cytometry analysis of FcεRI and c-Kit surface expression and purity of WT and CD151 −/− BMMC cultures. All data are representative of three independent experiments. Data are represented as mean ± SEM. * p

Article Snippet: Allophycocyanin-conjugated anti-mouse CD151 flow cytometry Ab was from R & D Systems (Minneapolis, MN).

Techniques: Cell Culture, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Negative Control, Mouse Assay, Staining

Journal: Scientific Reports

Article Title: TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells

doi: 10.1038/srep22040

Figure Lengend Snippet: MLN DC and LPDC take up and present CBir1 flagellin to T cells after mucosal challenge in vivo . Wild-type and TCRβ×δ −/− mice were gavaged with Alexa 647-labeled CBir1 flagellin. Uptake of CBir1 flagellin by MLN DC and LPDC was determined 2 hrs later by flow cytometry ( A ). When cultured with CD4 T cells of CBir1 Tg mice, LPDC from CBir1-gavaged TCRβxδ KO, but not wild-type, mice stimulated T cell proliferation ( B ). Data are reflective of 3 independent experiments. *p

Article Snippet: Antibodies and reagents Flow cytometry antibodies anti-mouse CD4-magnet beads were purchased from BD Bioscience.

Techniques: In Vivo, Mouse Assay, Labeling, Flow Cytometry, Cytometry, Cell Culture

Journal: Scientific Reports

Article Title: TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells

doi: 10.1038/srep22040

Figure Lengend Snippet: More LPDCs express TLR5 than spleen DCs and most CD172α + LPDCs are also TLR5 + . Spleen cells and LP cells were isolated from C57BL/6 mice. After depleting CD4 + T cells, CD4 − cells were stained with CD11c, CD11b, CD103, and TLR5 and analyzed by flow cytometry. ( A ) CD11c + cell expression of TLR5 was shown. ( B ) TLR5 expression by CD11b + and CD11b − DCs by gating on CD11c + population. ( C ) CD11c + DC expression of CD103 and CD11b by gating on CD11c + population. ( D ) Expression of TLR5 and CD172α by CD103 + DC and CD103 − DCs in LP of wild-type C57BL/6 mice by gating on CD11c + CD11b + population. ( E,F ) CD172α expression by CD103 + DC and CD103 − DCs in LP of wild-type ( E ) and TLR5 KO C57BL/6 mice ( F ) by gating on CD11c + population. FACS plots are reflective of 2–4 independent experiments.

Article Snippet: Antibodies and reagents Flow cytometry antibodies anti-mouse CD4-magnet beads were purchased from BD Bioscience.

Techniques: Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, FACS

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bone morphogenetic protein 9 enhances lipopolysaccharide-induced leukocyte recruitment to the vascular endothelium

doi: 10.4049/jimmunol.1601219

Figure Lengend Snippet: BMP6 increases surface expression of E-selectin, VCAM-1 and ICAM-1 on LPS-stimulated blood outgrowth endothelial cells (BOECs). BOECs were treated with BMP6 (16 h; 50 ng/ml) prior to the addition of LPS (4 h; 100 ng/ml). Flow cytometry was performed to

Article Snippet: Mouse monoclonal antibodies for flow cytometry: Anti-hE-selectin Fluorescein Conjugated mouse IgG1 (anti-human E-selectin-FITC; R & D Systems), Allophycocyanin (APC) mouse anti-human CD54 (anti-human ICAM-1-APC; BD Pharmingen), and PE/Cy5 anti-human CD106 (anti-human VCAM-1-PECy5; BioLegend).

Techniques: Expressing, Flow Cytometry, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Bone morphogenetic protein 9 enhances lipopolysaccharide-induced leukocyte recruitment to the vascular endothelium

doi: 10.4049/jimmunol.1601219

Figure Lengend Snippet: BMP9 increases surface expression of E-selectin and VCAM-1, but not ICAM-1 on LPS-stimulated ECs. ECs were treated with BMP9 (16 h; 5 ng/ml) prior to the addition of LPS (4 h; 100 ng/ml). Flow cytometry was performed to assess surface expression of E-selectin

Article Snippet: Mouse monoclonal antibodies for flow cytometry: Anti-hE-selectin Fluorescein Conjugated mouse IgG1 (anti-human E-selectin-FITC; R & D Systems), Allophycocyanin (APC) mouse anti-human CD54 (anti-human ICAM-1-APC; BD Pharmingen), and PE/Cy5 anti-human CD106 (anti-human VCAM-1-PECy5; BioLegend).

Techniques: Expressing, Flow Cytometry, Cytometry

Journal: Journal of Immunology Research

Article Title: Stabilized β-Catenin Ameliorates ALPS-Like Symptoms of B6/lpr Mice

doi: 10.1155/2017/3469108

Figure Lengend Snippet: β -cat Tg potentiated the apoptosis of T cells of lpr / lpr mice. WT, β -cat Tg , lpr / lpr , and β -cat Tg lpr / lpr female mice were sacrificed at age 8–12 weeks and splenocytes were isolated. The CD4 + T cells obtained from splenocytes were expanded in IL-2, IL-7, and IL-15 for 5 days. The T cell apoptosis then was induced by using different concentrations of anti-CD3 Ab ( μ g/ml). (a) The representative FACS results of T cells apoptosis with anti-CD3 Ab (10 μ g/ml). The cell pellets were stained with anti-CD3-APC, Annexin V, and 7-AAD surface markers, then the flow cytometry was used to analyze the apoptotic of T cells. (b) The representative statistical analysis of the apoptotic percentages of T cells. Data are shown as mean ± SD ( n = 3). ∗ p

Article Snippet: Antibodies and Flow Cytometry Anti-mouse CD3 (clone PC-61) (QuantoBio), anti-mouse TCRβ chain (clone H75-597) (eBioscience, San Diego, CA, USA), anti-mouse CD4 (clone RM4-5) (eBioscience), anti-mouse CD8a (clone 53-6.7) (eBioscience), rat anti-mouse CD44 (clone IM7) (BD Biosciences, Franklin Lakes, NJ, USA), and rat anti-mouse CD62L (MEL-14) (BD Biosciences) fluorochrome-labeled antibodies were used for flow cytometry.

Techniques: Mouse Assay, Isolation, FACS, Staining, Flow Cytometry, Cytometry

Journal: PLoS ONE

Article Title: Dengue immune sera enhance Zika virus infection in human peripheral blood monocytes through Fc gamma receptors

doi: 10.1371/journal.pone.0200478

Figure Lengend Snippet: Enhancement of ZIKV infection in primary monocytes triggered by DENV immune sera. Freshly isolated PBMCs from healthy donors were either uninfected or infected with ZIKV at an MOI of 2 in the presence or absence of serially diluted DENV immune serum samples. Cells were harvested and stained with anti-E antibody in combination with antibodies to CD3, CD14, CD19 and then analyzed by flow cytometry. (A) and (C) Representative flow cytometry analyses of ZIKV-infected (Anti-E) monocytes (CD14+CD3-CD19-), T cells (CD14-CD3+CD19-) and B cells (CD14-CD3-CD19+) in the presence of a DENV immune serum sample D1-E01 (A) and a control serum Naïve-01 (C) were presented. (B) and (D) Verification of enhancement in ZIKV infection by the boosted assay on Vero cells. Culture supernatants from ZIKV-infected PBMCs were harvested and used to infect Vero cells for two days followed by intracellular staining with an anti-E antibody and flow cytometry analysis. Representative results of the boosted infection assay on Vero cells for the DENV immune serum D1-01 (B) and the control serum Naïve-01 (D) were shown.

Article Snippet: Flow cytometry analysis Mouse anti-human antibodies anti-CD3 (PE-Cy7; clone UCHT1; Biolegend), anti-CD4 (BV-785; clone RPA-T4; Biolegend), anti-CD14 (PE; clone M5E2; Biolegend), anti-CD19 (BV-711; clone HIB19; Biolegend), anti-CD19 (APC; clone HIB19; Biolegend), anti-HLA-DR (Percp; clone L243; Biolegend), anti-CD11c (APC; clone 3.9; eBioscience), anti-DC-SIGN (PE-Cy7; clone 9E9A8; Biolegend), anti-CD83 (FITC; clone HB15e; Biolegend) and anti-CD86 (PE; clone IT2.2; eBioscience) were purchased from Biolegend and eBioscience.

Techniques: Infection, Isolation, Staining, Flow Cytometry, Cytometry

Journal: PLoS ONE

Article Title: Treg Cell Resistance to Apoptosis in DNA Vaccination for Experimental Autoimmune Encephalomyelitis Treatment

doi: 10.1371/journal.pone.0049994

Figure Lengend Snippet: Tolerogenic DCs were stimulated in treated EAE mice. On day 3 after the second treatment, the splenocytes were prepared and intracellularly stained with anti-CD11c and anti-IL-10 mAbs. A. CD11c + cells were counted relatively to total splenocytes by flow cytometry. B. CD11c + IL-10 + cells were counted relatively to total CD11c + DC cells by flow cytometry. Shown in each panel is 1 of at least 3 experiments with similar results.

Article Snippet: FK506 was from Astellas Ireland Co., Ltd. All antibodies for flow cytometry analysis were from eBioscience.

Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry

Journal: PLoS ONE

Article Title: Treg Cell Resistance to Apoptosis in DNA Vaccination for Experimental Autoimmune Encephalomyelitis Treatment

doi: 10.1371/journal.pone.0049994

Figure Lengend Snippet: Immune tolerance was restored in treated EAE mice. A. At day 4 after the second treatment, T cell proliferation by MTT method was performed with MOG 35–55 peptide as stimulation antigen, ConA as positive control and BSA as irrelevant antigen. B. At day 4 after the second treatment, the splenocytes of treated EAE mice were prepared for Treg cells analysis. Gating on CD4 + T cells, Treg cells (CD4 + CD25 + Foxp3 + ) were counted relatively to total CD4 cells by flow cytometry. C. Treg cells from naïve mice (nTreg) or FK506/p2MOG 35 treated mice (iTreg) were co-culture with CD4 + CD25 − T cells from naïve C57BL/6 mice respectively. Proliferation was tested by MTT method. Bar, mean and SD from 3 independent experiments, each using at least three mice per group (n = 3); *, p

Article Snippet: FK506 was from Astellas Ireland Co., Ltd. All antibodies for flow cytometry analysis were from eBioscience.

Techniques: Mouse Assay, MTT Assay, Positive Control, Flow Cytometry, Cytometry, Co-Culture Assay

Journal: PLoS ONE

Article Title: Treg Cell Resistance to Apoptosis in DNA Vaccination for Experimental Autoimmune Encephalomyelitis Treatment

doi: 10.1371/journal.pone.0049994

Figure Lengend Snippet: Suppression of Th1 and Th17 cell responses in treated EAE mice. At day 4 after the second treatment, the splenocytes of treated mice were prepared for intracellular staining. A. The splenocytes were intracellularly stained with anti-CD4 and anti-IFN-γ mAbs. CD4 + IFN-γ + T cells were counted relatively to total CD4 T cells by flow cytometry. B. The splenocytes were intracellularly stained with anti-CD4 and anti-IL-4 mAbs. CD4 + IL-4 + T cells were counted relatively to total CD4 T cells by flow cytometry. C. The splenocytes were intracellularly stained with anti-CD4 and anti-IL-17 mAbs. CD4 + IL-17 + T cells were counted relatively to total CD4 T cells by flow cytometry. Bar, mean and SD from 3 independent experiments, each using at least three mice per group (n = 3); *, p

Article Snippet: FK506 was from Astellas Ireland Co., Ltd. All antibodies for flow cytometry analysis were from eBioscience.

Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry

Journal: PLoS ONE

Article Title: Treg Cell Resistance to Apoptosis in DNA Vaccination for Experimental Autoimmune Encephalomyelitis Treatment

doi: 10.1371/journal.pone.0049994

Figure Lengend Snippet: CD4 T cells augmented apoptosis and Treg cells resisted apoptosis in treated EAE mice. A. On day 4 after the second treatment, the splenocytes were prepared for apoptosis test. A. The splenocytes gating on CD4 + T cells were analyzed for annexinV/PI by flow cytometry. Shown in each panel is 1 of at least 3 experiments with similar results. B. Gating on CD4 + CD25 + Treg cells, the samples were analyzed for apoptosis by flow cytometry. Shown in each panel is 1 of at least 3 experiments with similar results. C. Summary of the percentage of apoptotic and necrotic CD4 + T cells and Treg cells. Bar, mean and SD from 3 independent experiments, each using at least three mice per group (n = 3); *, p

Article Snippet: FK506 was from Astellas Ireland Co., Ltd. All antibodies for flow cytometry analysis were from eBioscience.

Techniques: Mouse Assay, Flow Cytometry, Cytometry

Journal: Haematologica

Article Title: Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression

doi: 10.3324/haematol.2016.154062

Figure Lengend Snippet: Resistin increases the expression of ABC transporters in myeloma cells. ARP-1 and MM.1S myeloma cells were cultured with resistin (50 ng/mL) for 12 h. Some of the cultured cells were labeled with eFluxx-ID gold fluorescent dye and further analyzed by flow cytometry. Others were subjected to RNA or protein extraction for real-time PCR or western blot analysis. (A) Intracellular eFluxx-ID gold fluorescence intensity was quantified. PBS, phosphate-buffered saline solution (controls). (B) Real-time PCR shows relative mRNA expression of ABC transporter genes. (C) Western blot analysis shows expression of ABCG2 and ABCC5 proteins. Cells cultured without resistin served as controls. GAPDH served as a protein loading control. (D) Real-time PCR analysis shows relative expression levels of ABCC5 and ABCG2 mRNA in ARP-1 or MM.1S cells bearing non-targeted siRNA ( si Ctrl) or the pooled siRNA of ABCC5 ( si C5) and ABCG2 ( si G2). (E) The percentages of apoptotic cells in si Ctrl- or both si C5- and si G2-expressing ARP-1 or MM.1S cells treated with or without resistin or melphalan (Mel) are shown. Results are representative of three independent experiments. * P

Article Snippet: Except where specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), all antibodies for flow cytometry analysis were purchased from BD Biosciences (San Jose, CA, USA), and all antibodies for western blot analysis were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Cell Culture, Labeling, Flow Cytometry, Cytometry, Protein Extraction, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence

Journal: Haematologica

Article Title: Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression

doi: 10.3324/haematol.2016.154062

Figure Lengend Snippet: Resistin protects myeloma from chemotherapy in vivo . SCID mice were injected with ARP-1 myeloma cells (5×10 5 cells per mouse) directly into the femur (n=5 mice per group). Three weeks after ARP-1 cell injection, mice began intraperitoneal treatment with melphalan (Mel; 50 μg/mouse), resistin (20 μg/mouse), or both every 3 days for 3 weeks. After treatment, the mouse sera were subjected to enzyme-linked immusorbent assay to measure M-protein levels. After the mice had been euthanized, the cells flushed from each mouse’s femoral bone marrow cavity were labeled with an antibody against human CD138, and the CD138 + cells were sorted by flow cytometry. CD138 + cells were subjected to an annexin V binding assay to determine cell apoptosis. The mouse femora were analyzed with an in situ TUNEL assay. Mice that received neither melphalan nor resistin served as controls. (A) Relative levels of M-proteins. (B) Percentages of CD138 + cells. (C) Percentages of apoptotic CD138 + cells. (D) Representative images of TUNEL + cells in bone marrow. (E) Quantitative analysis of TUNEL staining. Bar: 20 μm. Original magnification × 200. The results shown represent averages ± SD (n = 5 mice/group, 3 replicate studies). * P

Article Snippet: Except where specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), all antibodies for flow cytometry analysis were purchased from BD Biosciences (San Jose, CA, USA), and all antibodies for western blot analysis were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vivo, Mouse Assay, Injection, Labeling, Flow Cytometry, Cytometry, Binding Assay, In Situ, TUNEL Assay, Staining