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BioLegend pe labeled anti murine cd8
Transfecting bystander cells leads to activation of antigen-specific naïve <t>CD8</t> + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments
Pe Labeled Anti Murine Cd8, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity"

Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity

Journal: Journal of Controlled Release

doi: 10.1016/j.jconrel.2011.08.037

Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments
Figure Legend Snippet: Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments

Techniques Used: Activation Assay, Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay

Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and
Figure Legend Snippet: Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and

Techniques Used: Co-Culture Assay

Related Articles

other:

Article Title: Circulating myeloid cells invade the central nervous system to mediate cachexia during pancreatic cancer
Article Snippet: The following anti-mouse antibodies were used, with clone, fluorophore, and dilution indicated in parenthesis: CD3 (17A2, PE, 1:100), CD3 (17A2, APC/Cy7, 1:400), CD4 (RM4-5, APC, 1:100), CD8 (53–6.7, APC/Cy7, 1:800), CD11b (M1/70, APC, 1:800), CD11b (M1/70, FITC, 1:200), CD19 (6D5,BV650, 1:33), CD45 (30-F11, PerCP/Cy5.5, 1:400), CD45 (30-F11, APC/Cy7, 1:400), CX3CR1 (SAO11F11, PE/Cy7, 1:100), Dec205 (NLDC-145, APC,1:100), CD115 (AFS98, APC/CY7, 1:400), Ly6C (HK1.4, PerCP, 1:100), Ly6G (1A8, PE/Cy7, 1:800), NK1.1 (PK136, BV785, 1:800), CCR2 (SA203G11, AF647, 1:100).

Flow Cytometry:

Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes
Article Snippet: .. The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69). ..

Article Title: Arthritis Flares Mediated by Tissue Resident Memory T Cells in the Joint
Article Snippet: .. The following antibodies were used for the analysis of murine synovium with flow cytometry or cell sorting: Alexa Fluor 700® anti-CD3 (Clone 17A2, BioLegend Cat# 100216, RRID:AB_493697), APC anti-CD3 (Clone 17A2, BioLegend Cat# 100236, RRID:AB_2561456), FITC anti-CD45 (Clone 30-F11, BD Pharmingen Cat# 561088), FITC anti-CD45.1 (Clone A20, BioLegend Cat# 110706, RRID:AB_313495), PE anti-CD45.2 (Clone 104, BioLegend Cat# 109808, RRID:AB_313445), FITC anti-CD4 (Clone GK15, BioLegend Cat# 100406, RRID:AB_312691), APC/Cyanine7 anti-CD4 (Clone GK1.5, BioLegend Cat# 100414, RRID:AB_312699), Brilliant Violet™ 650 anti-CD8a (Clone 53-6.7, (BioLegend Cat# 100742, RRID:AB_2563056), PerCP/Cyanine5.5 anti-CD8a (Clone 53-6.7, BioLegend Cat# 100734, RRID:AB_2075238), Brilliant Violet™ 605 anti-CD44 (Clone IM7, BioLegend Cat# 103047, RRID:AB_2562451), APC anti-CD62L (Clone MEL-14, BioLegend Cat# 104412, RRID:AB_313099), Pacific Blue™ anti-CD69 (Clone H1.2F3, BioLegend Cat# 104524, RRID:AB_2074979), PE/Cyanine7 anti-CD103 (Clone 2E7, BioLegend Cat# 121426, RRID:AB_2563691), PE anti-CD103 (Clone 2E7, BioLegend Cat# 121406, RRID:AB_1133989), PE anti-ST2 (Clone DIH4, BioLegend Cat# 146608, RRID:AB_2728177), PE anti-CD19 (Clone eBio1D3, Thermo Fisher Scientific Cat# 12-0193-82, RRID:AB_657659), APC anti-CD11b (Clone M1/70, BioLegend Cat# 101212, RRID:AB_312795), Brilliant Violet™ 650 anti-Ly6G (Clone 1A8, BioLegend Cat# 127641, RRID:AB_2565881), Brilliant Violet™ 421 anti-NK1.1 (Clone PK136, BioLegend Cat# 108731, RRID:AB_10895916). ..

Cytometry:

Article Title: Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes
Article Snippet: .. The following anti-human antibodies were used for flow cytometry assays: CD3 (clone SK7) phycoerythrin (PE)-Cy7, CD4 (clone DE54), CD8 (clone SK1), CD45RA FITC, CD62L allophycocyanin, IFN-γ Alexa Fluor 647, granzyme B (clone GB11) A647, CD107a (clone H4A3) fluorescein isothiocyanate (FITC), CD107b (clone H4B4) FITC (BioLegend, San Diego, CA), and granzyme K (clone G3H69). ..

Staining:

Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity
Article Snippet: .. An aliquot of cells was stained with PE-labeled anti-murine CD8 (BioLegend) and PerCP Cy 5.5-labeled anti-murine CD90 (CD90.1) (BioLegend). ..

FACS:

Article Title: Arthritis Flares Mediated by Tissue Resident Memory T Cells in the Joint
Article Snippet: .. The following antibodies were used for the analysis of murine synovium with flow cytometry or cell sorting: Alexa Fluor 700® anti-CD3 (Clone 17A2, BioLegend Cat# 100216, RRID:AB_493697), APC anti-CD3 (Clone 17A2, BioLegend Cat# 100236, RRID:AB_2561456), FITC anti-CD45 (Clone 30-F11, BD Pharmingen Cat# 561088), FITC anti-CD45.1 (Clone A20, BioLegend Cat# 110706, RRID:AB_313495), PE anti-CD45.2 (Clone 104, BioLegend Cat# 109808, RRID:AB_313445), FITC anti-CD4 (Clone GK15, BioLegend Cat# 100406, RRID:AB_312691), APC/Cyanine7 anti-CD4 (Clone GK1.5, BioLegend Cat# 100414, RRID:AB_312699), Brilliant Violet™ 650 anti-CD8a (Clone 53-6.7, (BioLegend Cat# 100742, RRID:AB_2563056), PerCP/Cyanine5.5 anti-CD8a (Clone 53-6.7, BioLegend Cat# 100734, RRID:AB_2075238), Brilliant Violet™ 605 anti-CD44 (Clone IM7, BioLegend Cat# 103047, RRID:AB_2562451), APC anti-CD62L (Clone MEL-14, BioLegend Cat# 104412, RRID:AB_313099), Pacific Blue™ anti-CD69 (Clone H1.2F3, BioLegend Cat# 104524, RRID:AB_2074979), PE/Cyanine7 anti-CD103 (Clone 2E7, BioLegend Cat# 121426, RRID:AB_2563691), PE anti-CD103 (Clone 2E7, BioLegend Cat# 121406, RRID:AB_1133989), PE anti-ST2 (Clone DIH4, BioLegend Cat# 146608, RRID:AB_2728177), PE anti-CD19 (Clone eBio1D3, Thermo Fisher Scientific Cat# 12-0193-82, RRID:AB_657659), APC anti-CD11b (Clone M1/70, BioLegend Cat# 101212, RRID:AB_312795), Brilliant Violet™ 650 anti-Ly6G (Clone 1A8, BioLegend Cat# 127641, RRID:AB_2565881), Brilliant Violet™ 421 anti-NK1.1 (Clone PK136, BioLegend Cat# 108731, RRID:AB_10895916). ..

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    BioLegend pe cy5 labelled anti murine cd45r b220
    Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and <t>CD45R/B220</t> (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.
    Pe Cy5 Labelled Anti Murine Cd45r B220, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend anti mouse ifnγ antibodies
    Drp1 Controls the Metabolic Reprogramming of Activated T Cells (A) Mitochondria (TOM20) distribution in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated with anti-CD3-coated beads (referred to as B, labeled with anti-CD3 antibody, red) (n = 4). (B) Fluo3-AM-loaded +/+ cre+ control and fl/fl cre+ Drp1 KO T cells were incubated with the aCD3 antibody. After acquiring Fluo3-AM baseline fluorescence, a secondary antibody was added, and fluorescence was acquired up to 6 min. The fold increase in maximum (at 2 min) and residual (at 5 min) Fluo3-AM fluorescence relative to baseline is reported in the graph on the right (n = 5 ctrl, 4 KO). (C) Expression levels of the indicated (phospho)-protein in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated in vitro for the indicated time. Quantification of the KO:ctrl ratio for the indicated (phospho)-proteins is reported in the graph on the right (AMPK-mTOR, n = 5; cMyc, n = 4; S6, n = 3). cMyc levels are reported 48 hr post-stimulation (maximal upregulation), but similar results were also obtained at 5 hr. (D and E) Expression levels and relative quantifications of the indicated (phospho)-protein from +/+ cre+ control and fl/fl cre+ Drp1 KO T cells activated in vitro for 5 hr in the presence of the calcium chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EDTA (D, n = 3) or the AMPK inhibitor Compound-C (E, n = 3). (F and G) RNA sequencing (RNA-seq) analysis in 3-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO T cells. (F) Heatmap of cMyc-dependent metabolic genes in T cells (cMyc-MG) expression in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells, with glycolytic genes highlighted. F). TCA, tricarboxylic acid; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; FAO, fatty acid oxidation. For each group, transcriptional enrichment in KO cells compared with controls is highlighted in red, downregulation in blue and no net difference in black (n = 3). (H–J) Seahorse analysis of extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I and J) rates in 6-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+ T cells (2-DG, 2-deoxyglucose; Rot/an, rotenone and antimycin). FA oxidation was measured with BSA-palmitate with or without etomoxir (J). The following parameters were quantified: glycolysis (Glyc), maximal glycolytic capacity (MGC); basal OXPHOS (basal OX), maximum respiratory capacity (MRC), and basal (basal) and maximal (max) FA oxidation (n = 3). (K) MFI for IL7Ra (n = 17), CD44 (n = 12), KLRG1 (n = 9), <t>IFNγ</t> (n = 10), TNF-α (n = 6), IL-2 (n = 4), and IL-4 (n = 7) and for the Tbet:Eomes ratio (n = 8) in 6-day in vitro -activated CD8+ +/+ cre+ control and fl/fl cre+ Drp1 KO T cells under the indicated polarizing conditions. Data are represented as mean ± SEM. Scale bar, 10 μm in (A). Significance is indicated as follows: ∗ p
    Anti Mouse Ifnγ Antibodies, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse ifnγ antibodies/product/BioLegend
    Average 99 stars, based on 1 article reviews
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    99
    BioLegend pe labeled anti murine cd8
    Transfecting bystander cells leads to activation of antigen-specific naïve <t>CD8</t> + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments
    Pe Labeled Anti Murine Cd8, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe labeled anti murine cd8/product/BioLegend
    Average 99 stars, based on 1 article reviews
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    pe labeled anti murine cd8 - by Bioz Stars, 2021-09
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    97
    BioLegend pe labelled anti ifnγ
    SLAM co-stimulation promotes Th17 differentiation in naïve CD4 + T cells (A)-(C) Naïve (CD45RA + CD62L hi ) CD4 + T cells were stimulated with anti-CD3 and co-stimulatory anti-CD28, anti-SLAMF3, SLAMF6 and control IgG under Th17 conditions for 7 days. IL-17 and <t>IFNγ</t> production was analyzed by intracellular staining and ELISA. (A) Secretion of IL-17 by naïve CD4 + T cells on day 7 was measured by ELISA. (B) shows percentages of IL-17 producing cells. The relative number of IL-17 producing cells is given as a percentage of naïve CD4 + T cells. (C) One representative experiment is displayed (from day 7). (D) Kinetics of IL-17 secretion by naïve CD4 + T cells in response to co-stimulation through CD28, SLAMF3 and SLAMF6. On days 3, 5 and 7 the IL-17 levels were monitored by ELISA. (E) Naïve CD4 + T cells were labeled with CFSE, then stimulated and differentiated until day 7. Staining on day 7 was as above. No differences in CFSE staining were detected between the different groups, indicating comparable proliferation rates.
    Pe Labelled Anti Ifnγ, supplied by BioLegend, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.

    Journal: Journal of Immunology Research

    Article Title: OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics

    doi: 10.1155/2018/2505818

    Figure Lengend Snippet: Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.

    Article Snippet: Murine blood cells were stained with FITC-labelled anti-murine CD19 (Biolegend, ImTec Diagnostics N.V., Antwerp, Belgium) and PE/Cy5-labelled anti-murine CD45R/B220 (Biolegend, ImTec Diagnostics N.V., Antwerp, Belgium) and analyzed with the 3-color Becton Dickinson FACSCalibur apparatus.

    Techniques: Mouse Assay, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot, Amplification, Agarose Gel Electrophoresis, Staining, Knock-Out, Expressing

    Drp1 Controls the Metabolic Reprogramming of Activated T Cells (A) Mitochondria (TOM20) distribution in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated with anti-CD3-coated beads (referred to as B, labeled with anti-CD3 antibody, red) (n = 4). (B) Fluo3-AM-loaded +/+ cre+ control and fl/fl cre+ Drp1 KO T cells were incubated with the aCD3 antibody. After acquiring Fluo3-AM baseline fluorescence, a secondary antibody was added, and fluorescence was acquired up to 6 min. The fold increase in maximum (at 2 min) and residual (at 5 min) Fluo3-AM fluorescence relative to baseline is reported in the graph on the right (n = 5 ctrl, 4 KO). (C) Expression levels of the indicated (phospho)-protein in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated in vitro for the indicated time. Quantification of the KO:ctrl ratio for the indicated (phospho)-proteins is reported in the graph on the right (AMPK-mTOR, n = 5; cMyc, n = 4; S6, n = 3). cMyc levels are reported 48 hr post-stimulation (maximal upregulation), but similar results were also obtained at 5 hr. (D and E) Expression levels and relative quantifications of the indicated (phospho)-protein from +/+ cre+ control and fl/fl cre+ Drp1 KO T cells activated in vitro for 5 hr in the presence of the calcium chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EDTA (D, n = 3) or the AMPK inhibitor Compound-C (E, n = 3). (F and G) RNA sequencing (RNA-seq) analysis in 3-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO T cells. (F) Heatmap of cMyc-dependent metabolic genes in T cells (cMyc-MG) expression in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells, with glycolytic genes highlighted. F). TCA, tricarboxylic acid; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; FAO, fatty acid oxidation. For each group, transcriptional enrichment in KO cells compared with controls is highlighted in red, downregulation in blue and no net difference in black (n = 3). (H–J) Seahorse analysis of extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I and J) rates in 6-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+ T cells (2-DG, 2-deoxyglucose; Rot/an, rotenone and antimycin). FA oxidation was measured with BSA-palmitate with or without etomoxir (J). The following parameters were quantified: glycolysis (Glyc), maximal glycolytic capacity (MGC); basal OXPHOS (basal OX), maximum respiratory capacity (MRC), and basal (basal) and maximal (max) FA oxidation (n = 3). (K) MFI for IL7Ra (n = 17), CD44 (n = 12), KLRG1 (n = 9), IFNγ (n = 10), TNF-α (n = 6), IL-2 (n = 4), and IL-4 (n = 7) and for the Tbet:Eomes ratio (n = 8) in 6-day in vitro -activated CD8+ +/+ cre+ control and fl/fl cre+ Drp1 KO T cells under the indicated polarizing conditions. Data are represented as mean ± SEM. Scale bar, 10 μm in (A). Significance is indicated as follows: ∗ p

    Journal: Cell Reports

    Article Title: Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming

    doi: 10.1016/j.celrep.2018.11.018

    Figure Lengend Snippet: Drp1 Controls the Metabolic Reprogramming of Activated T Cells (A) Mitochondria (TOM20) distribution in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated with anti-CD3-coated beads (referred to as B, labeled with anti-CD3 antibody, red) (n = 4). (B) Fluo3-AM-loaded +/+ cre+ control and fl/fl cre+ Drp1 KO T cells were incubated with the aCD3 antibody. After acquiring Fluo3-AM baseline fluorescence, a secondary antibody was added, and fluorescence was acquired up to 6 min. The fold increase in maximum (at 2 min) and residual (at 5 min) Fluo3-AM fluorescence relative to baseline is reported in the graph on the right (n = 5 ctrl, 4 KO). (C) Expression levels of the indicated (phospho)-protein in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated in vitro for the indicated time. Quantification of the KO:ctrl ratio for the indicated (phospho)-proteins is reported in the graph on the right (AMPK-mTOR, n = 5; cMyc, n = 4; S6, n = 3). cMyc levels are reported 48 hr post-stimulation (maximal upregulation), but similar results were also obtained at 5 hr. (D and E) Expression levels and relative quantifications of the indicated (phospho)-protein from +/+ cre+ control and fl/fl cre+ Drp1 KO T cells activated in vitro for 5 hr in the presence of the calcium chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EDTA (D, n = 3) or the AMPK inhibitor Compound-C (E, n = 3). (F and G) RNA sequencing (RNA-seq) analysis in 3-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO T cells. (F) Heatmap of cMyc-dependent metabolic genes in T cells (cMyc-MG) expression in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells, with glycolytic genes highlighted. F). TCA, tricarboxylic acid; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; FAO, fatty acid oxidation. For each group, transcriptional enrichment in KO cells compared with controls is highlighted in red, downregulation in blue and no net difference in black (n = 3). (H–J) Seahorse analysis of extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I and J) rates in 6-day in vitro -activated +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+ T cells (2-DG, 2-deoxyglucose; Rot/an, rotenone and antimycin). FA oxidation was measured with BSA-palmitate with or without etomoxir (J). The following parameters were quantified: glycolysis (Glyc), maximal glycolytic capacity (MGC); basal OXPHOS (basal OX), maximum respiratory capacity (MRC), and basal (basal) and maximal (max) FA oxidation (n = 3). (K) MFI for IL7Ra (n = 17), CD44 (n = 12), KLRG1 (n = 9), IFNγ (n = 10), TNF-α (n = 6), IL-2 (n = 4), and IL-4 (n = 7) and for the Tbet:Eomes ratio (n = 8) in 6-day in vitro -activated CD8+ +/+ cre+ control and fl/fl cre+ Drp1 KO T cells under the indicated polarizing conditions. Data are represented as mean ± SEM. Scale bar, 10 μm in (A). Significance is indicated as follows: ∗ p

    Article Snippet: For evaluation of IL4 in CD4+ T, 100ng/ml mouse IL4 (R & D System) and 10μg/ml anti-mouse-IL12 and anti-mouse IFNγ antibodies (Biolegend) have been added to the cells all the days (th2 polarizing conditions).

    Techniques: Labeling, Incubation, Fluorescence, Expressing, In Vitro, RNA Sequencing Assay

    Drp1 Is Required for T Cell Accumulation into Draining LNs and for Infiltration and Exhaustion into Tumor Masses during Immune-Surveillance (A and B) In vitro -activated control (eFluor670-labeled) and Drp1 KO (CFSE-labeled) T cells were injected i.v. into WT recipient mice bearing 13 day-old MC38-derived tumors. After 24 hr, peripheral blood (PB), inguinal DLNs, and tumor masses (TILs) were collected, and the KO:control ratio (A, both total T cells or CD8+ T cells only) and the exogenous CD8+:CD4+ T cell ratio (B) (considering both control and KO cells) were quantified by flow cytometry (TILs, n = 4; DLNs, n = 6). (C) Size of MC38-induced s.c. tumors in +/+ cre+ control and fl/fl cre+ Drp1 conditional KO mice at the indicated times (n = 11 ctrl, 8 KO). (D) Tumor weight graphs and pictures of representative MC38-derived tumors isolated 18 days after cell injection (n = 11 ctrl, 8 KO). (E and F) Absolute number of +/+ cre+ control and fl/fl cre+ Drp1 KO CD4+ and CD8+ T cells collected from inguinal DLNs (E) or from MC38-derived tumors (TILs) (F) 18 days after s.c. tumor cell injection (n = 14 ctrl, 11 KO). (G) CD8+ or CD4+ IHC staining on 18-day-grown isolated MC38-derived tumor slices. Quantification of CD8+ and CD4+ TILs density is reported in the corresponding graph (n = 5 ctrl, 6 KO). (H) Density of dextramer + CD8+ TILs per field, calculated by combining the percentage of recovered CD8+ dextramer + cells by cytofluorimetric analysis with the quantification of total CD8+ TIL density by IHC (n = 5 ctrl, 5 KO). (I) Correlation between tumor size and CD8+ TIL percentage among CD45+ cells in tumor-bearing +/+ cre+ control and fl/fl cre+ Drp1 KO mice. R = −0.661, p = 0.019 (n = 5 ctrl, 7 KO). (J and K) Relative expression of CX3CR1 (negatively, intermediately, or highly expressed) (J, n = 9) or PD1 (K, n = 9) in +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+CD44+ T cells collected from DLNs or from MC38-derived tumors (TILs) 18 days after s.c. tumor cell injection. (L) Percentage of CD44+ cells among all CD8+ and of IFNγ+, Tbet+, and Eomes+ cells among all CD8+CD44+ T cells isolated from DLNs or in the tumors (TILs) of +/+ cre+ control and fl/fl cre+ Drp1 KO mice 18 days after s.c. tumor cell injection and after 6 hr of in vitro T cell re-stimulation (n = 5 ctrl, 7 KO). Data are represented as mean ± SEM in (C), (E), and (J) and as dot density plots in (A), (B), (D), (F)–(H), (K), and (L). Data are from two representative of four independent experiments. Scale bars, 5 mm in (D) and 100 μm in (G). Significance is indicated as follows: ∗ p

    Journal: Cell Reports

    Article Title: Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming

    doi: 10.1016/j.celrep.2018.11.018

    Figure Lengend Snippet: Drp1 Is Required for T Cell Accumulation into Draining LNs and for Infiltration and Exhaustion into Tumor Masses during Immune-Surveillance (A and B) In vitro -activated control (eFluor670-labeled) and Drp1 KO (CFSE-labeled) T cells were injected i.v. into WT recipient mice bearing 13 day-old MC38-derived tumors. After 24 hr, peripheral blood (PB), inguinal DLNs, and tumor masses (TILs) were collected, and the KO:control ratio (A, both total T cells or CD8+ T cells only) and the exogenous CD8+:CD4+ T cell ratio (B) (considering both control and KO cells) were quantified by flow cytometry (TILs, n = 4; DLNs, n = 6). (C) Size of MC38-induced s.c. tumors in +/+ cre+ control and fl/fl cre+ Drp1 conditional KO mice at the indicated times (n = 11 ctrl, 8 KO). (D) Tumor weight graphs and pictures of representative MC38-derived tumors isolated 18 days after cell injection (n = 11 ctrl, 8 KO). (E and F) Absolute number of +/+ cre+ control and fl/fl cre+ Drp1 KO CD4+ and CD8+ T cells collected from inguinal DLNs (E) or from MC38-derived tumors (TILs) (F) 18 days after s.c. tumor cell injection (n = 14 ctrl, 11 KO). (G) CD8+ or CD4+ IHC staining on 18-day-grown isolated MC38-derived tumor slices. Quantification of CD8+ and CD4+ TILs density is reported in the corresponding graph (n = 5 ctrl, 6 KO). (H) Density of dextramer + CD8+ TILs per field, calculated by combining the percentage of recovered CD8+ dextramer + cells by cytofluorimetric analysis with the quantification of total CD8+ TIL density by IHC (n = 5 ctrl, 5 KO). (I) Correlation between tumor size and CD8+ TIL percentage among CD45+ cells in tumor-bearing +/+ cre+ control and fl/fl cre+ Drp1 KO mice. R = −0.661, p = 0.019 (n = 5 ctrl, 7 KO). (J and K) Relative expression of CX3CR1 (negatively, intermediately, or highly expressed) (J, n = 9) or PD1 (K, n = 9) in +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+CD44+ T cells collected from DLNs or from MC38-derived tumors (TILs) 18 days after s.c. tumor cell injection. (L) Percentage of CD44+ cells among all CD8+ and of IFNγ+, Tbet+, and Eomes+ cells among all CD8+CD44+ T cells isolated from DLNs or in the tumors (TILs) of +/+ cre+ control and fl/fl cre+ Drp1 KO mice 18 days after s.c. tumor cell injection and after 6 hr of in vitro T cell re-stimulation (n = 5 ctrl, 7 KO). Data are represented as mean ± SEM in (C), (E), and (J) and as dot density plots in (A), (B), (D), (F)–(H), (K), and (L). Data are from two representative of four independent experiments. Scale bars, 5 mm in (D) and 100 μm in (G). Significance is indicated as follows: ∗ p

    Article Snippet: For evaluation of IL4 in CD4+ T, 100ng/ml mouse IL4 (R & D System) and 10μg/ml anti-mouse-IL12 and anti-mouse IFNγ antibodies (Biolegend) have been added to the cells all the days (th2 polarizing conditions).

    Techniques: In Vitro, Labeling, Injection, Mouse Assay, Derivative Assay, Flow Cytometry, Cytometry, Isolation, Immunohistochemistry, Staining, Expressing

    Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments

    Journal: Journal of Controlled Release

    Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity

    doi: 10.1016/j.jconrel.2011.08.037

    Figure Lengend Snippet: Transfecting bystander cells leads to activation of antigen-specific naïve CD8 + T cells. SIINFEKL-specific OT-1 T cells were co-cultured with DCs and transfected fibroblasts and the amount of IFN-γ secretion was determined by ELISA. Experiments

    Article Snippet: An aliquot of cells was stained with PE-labeled anti-murine CD8 (BioLegend) and PerCP Cy 5.5-labeled anti-murine CD90 (CD90.1) (BioLegend).

    Techniques: Activation Assay, Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay

    Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and

    Journal: Journal of Controlled Release

    Article Title: Polymer-Mediated DNA Vaccine Delivery via Bystander Cells Requires a Proper Balance between Transfection Efficiency and Cytotoxicity

    doi: 10.1016/j.jconrel.2011.08.037

    Figure Lengend Snippet: Schematic illustration of a co-culture system for the study of polymer-mediated DNA vaccine delivery to bystander cells. The process involves bystander cells (fibroblasts), dendritic cells (DCs), and antigen-specific CD8 + T cells, mixed sequentially and

    Article Snippet: An aliquot of cells was stained with PE-labeled anti-murine CD8 (BioLegend) and PerCP Cy 5.5-labeled anti-murine CD90 (CD90.1) (BioLegend).

    Techniques: Co-Culture Assay

    SLAM co-stimulation promotes Th17 differentiation in naïve CD4 + T cells (A)-(C) Naïve (CD45RA + CD62L hi ) CD4 + T cells were stimulated with anti-CD3 and co-stimulatory anti-CD28, anti-SLAMF3, SLAMF6 and control IgG under Th17 conditions for 7 days. IL-17 and IFNγ production was analyzed by intracellular staining and ELISA. (A) Secretion of IL-17 by naïve CD4 + T cells on day 7 was measured by ELISA. (B) shows percentages of IL-17 producing cells. The relative number of IL-17 producing cells is given as a percentage of naïve CD4 + T cells. (C) One representative experiment is displayed (from day 7). (D) Kinetics of IL-17 secretion by naïve CD4 + T cells in response to co-stimulation through CD28, SLAMF3 and SLAMF6. On days 3, 5 and 7 the IL-17 levels were monitored by ELISA. (E) Naïve CD4 + T cells were labeled with CFSE, then stimulated and differentiated until day 7. Staining on day 7 was as above. No differences in CFSE staining were detected between the different groups, indicating comparable proliferation rates.

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

    Article Title: Increased expression of SLAM receptors SLAMF3 and SLAMF6 in systemic lupus erythematosus T lymphocytes promotes Th17 differentiation

    doi: 10.4049/jimmunol.1102773

    Figure Lengend Snippet: SLAM co-stimulation promotes Th17 differentiation in naïve CD4 + T cells (A)-(C) Naïve (CD45RA + CD62L hi ) CD4 + T cells were stimulated with anti-CD3 and co-stimulatory anti-CD28, anti-SLAMF3, SLAMF6 and control IgG under Th17 conditions for 7 days. IL-17 and IFNγ production was analyzed by intracellular staining and ELISA. (A) Secretion of IL-17 by naïve CD4 + T cells on day 7 was measured by ELISA. (B) shows percentages of IL-17 producing cells. The relative number of IL-17 producing cells is given as a percentage of naïve CD4 + T cells. (C) One representative experiment is displayed (from day 7). (D) Kinetics of IL-17 secretion by naïve CD4 + T cells in response to co-stimulation through CD28, SLAMF3 and SLAMF6. On days 3, 5 and 7 the IL-17 levels were monitored by ELISA. (E) Naïve CD4 + T cells were labeled with CFSE, then stimulated and differentiated until day 7. Staining on day 7 was as above. No differences in CFSE staining were detected between the different groups, indicating comparable proliferation rates.

    Article Snippet: Intracellular T cell staining for IL-17 and IFNγ was performed at the indicated time points using the BD Cytofix/Cytoperm Kit and Alexa-647-labelled anti-IL-17 (BD Biosciences) and PE-labelled anti-IFNγ (Biolegend) antibodies.

    Techniques: Staining, Enzyme-linked Immunosorbent Assay, Labeling