easysep mouse cd8+ t cell enrichment kit  (STEMCELL Technologies Inc)

Journal: Cells

Article Title: Dopaminergic Signalling Enhances IL-2 Production and Strengthens Anti-Tumour Response Exerted by Cytotoxic T Lymphocytes in a Melanoma Mouse Model

doi: 10.3390/cells11223536

Figure Lengend Snippet: Drd3 -deficiency results in attenuated CD8 + T-cell expansion and reduced IFN-γ production. Splenic CD8 + T-cells were isolated from Drd3 +/+ or Drd3 −/− OT-I mice, and then i.v. transferred into male or female C57BL/6 mice (2 × 10 5 cells/mouse). A group of mice did not receive the transfer of exogenous T-cells (Control). One day later, mice received an intradermal vaccination with 40μg of pVAX-OVA and, 11 days after vaccination, blood samples were obtained. ( A ) The expansion of OT-I cells was evaluated as the percentage of cells expressing the transgenic TCR (Vα2 Vβ5) in the CD3 + CD8 + ZAq − gate. Representative dot-plots are shown in the top panel. Numbers indicate the percentage of cells in the indicated region. Quantification is shown in the bottom panel. ( B ) Cells were left unstimulated or stimulated with pOT-I (1 μg/mL) for 6 h, and the intracellular production of IFNγ was evaluated in the CD8 + T-cell population by flow cytometry. Representative dot-plots are shown in the top panel. Numbers indicate the percentage of cells in the indicated region. Quantification of the percentage of IFNγ + CD8 + T-cells (left bottom panel) and the mean fluorescence intensity (MFI) associated with IFNγ + in the CD8 + ZAq − gate are shown. ( A , B ) Data from three independent experiments ( n = 15–18 mice per group) is shown. Each symbol represents data from an individual mouse. The mean ± SEM are depicted. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 by one-way ( A ) or two-way ( B ) ANOVA followed by Tukey’s post hoc test. n.s., not significant.

Article Snippet: Splenic CD8 + T-cells were purified from secondary lymphoid organs of Drd3 +/+ or Drd3 −/− OT-I mice using the EasySep Mouse CD8 + T-cell Enrichment Kit (StemCell Technologies; cat # 19853).

Techniques: Isolation, Control, Expressing, Transgenic Assay, Flow Cytometry, Fluorescence

Journal: Cells

Article Title: Dopaminergic Signalling Enhances IL-2 Production and Strengthens Anti-Tumour Response Exerted by Cytotoxic T Lymphocytes in a Melanoma Mouse Model

doi: 10.3390/cells11223536

Figure Lengend Snippet: Drd3 -deficiency does not affect CD8 + T-cell migration through secondary lymphoid organs. Splenic CD8 + T-cells were isolated from Drd3 +/+ Cd45.1 +/− CD45.2 +/− or Drd3 −/− Cd45.1 −/− CD45.2 +/+ OT-I mice and then mixed in a 1:1 ratio and i.v. transferred into Cd45.1 +/+ CD45.2 −/− male C57BL/6 mice (2 × 10 6 total cells/mouse). Twenty-four hours later, inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), and the spleen were collected, and CD8 + T-cells were analysed by flow cytometry. ( A ) A representative dot-plot of the input of 1:1 mixed donors’ cells is shown. ( B ) Representative dot-plots analysing the proportion of Drd3 +/+ (CD45.1 + CD45.2 + ) and Drd3 −/− (CD45.1 − CD45.2 + ) donor’s OT-I cells in the CD8 + ZAq − gate from different secondary lymphoid organs are shown. ( A,B ) Numbers indicate the percentage of cells in the indicated region. ( C ) Quantification of the percentage of Drd3 +/+ and Drd3 −/− donors’ OT-I cells in the CD8 + ZAq − gate. ( D , E ) Quantification of the MFI associated with CD62L ( D ) and CCR7 ( E ) expression on Drd3 +/+ and Drd3 −/− donors’ OT-I cells infiltrating different secondary lymphoid organs are shown. ( C – E ) Each symbol represents data from an individual mouse. Data from two independent experiments is shown. The mean ± SEM are depicted. No significant differences were found by two-way ANOVA.

Article Snippet: Splenic CD8 + T-cells were purified from secondary lymphoid organs of Drd3 +/+ or Drd3 −/− OT-I mice using the EasySep Mouse CD8 + T-cell Enrichment Kit (StemCell Technologies; cat # 19853).

Techniques: Migration, Isolation, Flow Cytometry, Expressing

Journal: Cells

Article Title: Dopaminergic Signalling Enhances IL-2 Production and Strengthens Anti-Tumour Response Exerted by Cytotoxic T Lymphocytes in a Melanoma Mouse Model

doi: 10.3390/cells11223536

Figure Lengend Snippet: DRD3-signalling strengthens IFN-γ production by favouring IL-2 release. ( A ) Splenic CD8 + T-cells were isolated from Drd3 +/+ or Drd3 −/− OT-I male mice and stimulated with anti-CD3 and anti-CD28 Abs in the absence of exogenous IL-2 for 48 h. Subsequently, cells were cultured for an additional 48 h in the absence of anti-CD3 and anti-CD28 Abs. The pOT-I was added to the cultures for the last 6 h, and then IL-2 was evaluated by intracellular immunostaining followed by flow cytometry analysis. The left panel shows representative histograms. The right panel shows the quantification. Values are the MFI associated with IL-2 in the CD8 + ZAq − gate normalised to the value of IL-2 MFI obtained for non-stimulated OT-I cells. ( B ) Splenic CD8 + T-cells isolated from Drd3 +/+ or Drd3 −/− male mice were non-treated or activated with anti-CD3 and anti-CD28 Abs in the absence of exogenous IL-2 for 24 h, and the extent of IL-2 secretion into the supernatant was quantified by ELISA. Each symbol represents data obtained from an individual mouse. ( C , D ) Drd3 +/+ or Drd3 −/− OT-I CD8 + T-cells were activated with anti-CD3 and anti-CD28 Abs in the absence or the presence of exogenous IL-2 during 48 h. Cells were cultured for an additional 48 h in the absence of anti-CD3 and anti-CD28 Abs. The pOT-I was added to the cultures for the last 6 h, and the intracellular IFNγ production ( C ) and surface CD25 expression ( D ) were assessed by flow cytometry. ( C , D ) The top panels show representative histograms and the bottom panels the quantification of the percentage of IFNγ + cells ( C ) and the normalised MFI for CD25 ( D ) in the CD8 + ZAq − gate. The histograms associated with autofluorescence are shown as a reference in the top panels. ( A – D ) Each symbol represents data obtained from a different mouse. Data from four ( A , B , D ) or seven ( C ) independent experiments is shown. The mean ± SEM are depicted. *, p < 0.05; **, p < 0.01; by Student’s t -test ( A ) or two-way ANOVA followed by Tukey’s post hoc test ( B , C ).

Article Snippet: Splenic CD8 + T-cells were purified from secondary lymphoid organs of Drd3 +/+ or Drd3 −/− OT-I mice using the EasySep Mouse CD8 + T-cell Enrichment Kit (StemCell Technologies; cat # 19853).

Techniques: Isolation, Cell Culture, Immunostaining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: Dopaminergic Signalling Enhances IL-2 Production and Strengthens Anti-Tumour Response Exerted by Cytotoxic T Lymphocytes in a Melanoma Mouse Model

doi: 10.3390/cells11223536

Figure Lengend Snippet: DRD3 stimulation reduces ERK1/2 phosphorylation and increases IL-2 production triggered by the TCR activation. ( A ) Splenic Drd3 +/+ or Drd3 −/− CD8 + T-cells were purified and then left unstimulated or stimulated with either anti-CD3 Ab, PD128907, or both together for 5 min. Afterwards, cells were lysed and the extent of ERK1/2-phosphorylation (pERK1/2; top panel) or total ERK1/2 (ERK1/2; bottom panel) were analysed by western blot. Results from a representative of three independent experiments are shown. (B-C) Drd3 +/+ OT-I CD8 + T-cells were left untreated or treated with 50 nM PD128907 for 30 min and then activated in the presence or absence of exogenous IL-2 (as described in .). The intracellular IL-2 production ( B ) and surface CD25 expression ( C ) were assessed by flow cytometry. Values are the MFI obtained in four independent experiments. Each symbol represents data obtained from a different mouse. Data from four independent experiments are shown. *, p < 0.05; **, p < 0.01; by paired Student’s t -test.

Article Snippet: Splenic CD8 + T-cells were purified from secondary lymphoid organs of Drd3 +/+ or Drd3 −/− OT-I mice using the EasySep Mouse CD8 + T-cell Enrichment Kit (StemCell Technologies; cat # 19853).

Techniques: Phospho-proteomics, Activation Assay, Purification, Western Blot, Expressing, Flow Cytometry

Journal: Cells

Article Title: Dopaminergic Signalling Enhances IL-2 Production and Strengthens Anti-Tumour Response Exerted by Cytotoxic T Lymphocytes in a Melanoma Mouse Model

doi: 10.3390/cells11223536

Figure Lengend Snippet: Drd3 -deficient CD8 + T-cells exhibit reduced potency in the anti-tumour response in male mice. Splenic CD8 + T-cells were isolated from Drd3 +/+ or Drd3 −/− OT-I mice, and then i.v. transferred into male C57BL/6 mice (2 × 10 5 cells/mouse). A group of mice did not receive the transfer of exogenous T-cells (Control). One day later, mice received an intradermal vaccination with 40 μg of pVAX-OVA. Thirteen days after vaccination, mice were s.c. inoculated with B16/OT-I melanoma cells (10 6 cells/mouse), and tumour growth and mice survival were monitored over time. ( A ) The experimental design is illustrated. ( B ) Tumour growth is represented as tumour volume in time. Data are the mean ± SEM from five mice per group. The top grey line indicates the frame of time where significant differences were found compared to the control group, and the red line indicates the frame of time where significant differences were found between mice receiving Drd3 +/+ or Drd3 −/− OT-I cells. ( C ) Tumour growth curves for individual mice are shown. ( D ) Mice’s survival over time is shown. Data from a representative (n = 5 mice per group) of two independent experiments are shown. *, p < 0.05; **, p < 0.01 by two-way ANOVA followed by Tukey’s post hoc test (B, grey line), by multiple t-test (B, red line) or by logrank test ( D ). n.s., not significant.

Article Snippet: Splenic CD8 + T-cells were purified from secondary lymphoid organs of Drd3 +/+ or Drd3 −/− OT-I mice using the EasySep Mouse CD8 + T-cell Enrichment Kit (StemCell Technologies; cat # 19853).

Techniques: Isolation, Control