Recombinant:Article Title: Impact of AIM2 on HNSCC Development
Article Snippet: Naïve CD4 + T cells and naïve CD8a + T cells were enriched using either a naïve CD4 isolation kit (Miltenyi Biotec, 130-104-453) or naïve CD8a isolation kit (Miltenyi Biotec, 130-096-543) and MACS separation columns (Miltenyi Biotec, 103-042-401). .. Then, 1 x 10 6 cells were stimulated in an αCD3 antibody (Invitrogen, 16-0031-coated plate at 2 μg/mL with 0.5μg/mL αCD28 antibody (Invitrogen, 16-0281-82), 5 ng/mL murine IL-2 (Peprotech, 212-12), 10 ng/mL recombinant mouse IL-12 (R&D Systems, 419-ML-010/CF) and 1 μg/mL αIL-4 (Invitrogen, 14-7041-85) for 3 days for CD4 cells. ..
Article Title: MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.
Article Snippet: In vitro activation of CD4+ T lymphocytes was achieved by incubation of cells on 10 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X Cell) in presence of 2 μg/ml anti-CD28 (clone 37.51, BD Biosciences). .. In vitro polarized TH1 lymphocytes were generated by adding 10 ng/ml recombinant mouse IL-12 (R&D Systems) and 20 μg/ml anti-IL-4 (clone 11B11, Bio X Cell) to the culture media during CD4+ T lymphocyte activation. .. In vitro polarized TH17 lymphocytes were generated with the addition of 20 ng/ml recombinant mouse IL-6 (R&D Systems), 20 ng/ml recombinant mouse IL-23 (R&D Systems), 4 ng/ml recombinant human (rh) TGFβ (R&D Systems) and 20 μg/ml anti-IFNγ (clone XMG1.2, Bio X Cell).
Article Title: Autotaxin in encephalitogenic CD4 T cells as a therapeutic target for multiple sclerosis.
Article Snippet: T cells were plated in 48-well plates at 1 × 106 cells/well and activated with Dynabeads Mouse T-Activator CD3/CD28 beads (Gibco) at 1:10 (beads:cells). .. Recombinant mouse IL-12 (R&D Systems) and recombinant mouse IL-23 (R&D Systems) were added to the culture at 0.5 and 5 ng/mL, respectively, and cells were incubated for 48 h. Samples were collected, washed once, and replated in serum-free media for 24 h with either vehicle or HA-130 (1 μM). .. Supernatants were then collected, and an autotaxin activity assay (Eschelon Bioscience) was run.
Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity
Article Snippet: .. The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302). .. Mice C57BL/6 mice were purchased from Shanghai Model Organisms Center, Inc., and bred in our facility under specific pathogen-free conditions.
Article Title: A strategy for uncovering germline variants altering anti-tumor CD8 T cell response.
Article Snippet: Among many factors known to alter the outcomes of T cell receptor (TCR)-induced proximal signaling, the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address.. Here, we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations (pTyr-SNVs) that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways.. We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin (OVA) antigens and retrovirally engineered syngeneic major histocompatibility complex (MHC) Class I restricted OVA TCR-transgenic CD8 T cells (OT-I).
Article Title: Trabectedin enhances the antitumor effects of IL-12 in triple-negative breast cancer
Article Snippet: IL-12 is a potent NK cell–stimulating cytokine, but the presence of immunosuppressive myeloid cells such as myeloid-derived suppressor cells (MDSC) can inhibit IL-12–induced NK-cell cytotoxicity.. Thus, we hypothesized that trabectedin, a myeloid cell– depleting agent, would improve the efficacy of IL-12 in triplenegative breast cancer (TNBC).. In vitro treatment of healthy donor NK cells with trabectedin increased expression of the activation marker CD69 and mRNA expression of T-box transcription factor (Tbx21), the cytotoxic ligands TNF-related apoptosis–inducing ligand (TNFSF10), Fas ligand (FASLG), and the dendritic cell (DC)– recruiting chemokine lymphotactin (XCL1).
In Vitro:Article Title: MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.
Article Snippet: In vitro activation of CD4+ T lymphocytes was achieved by incubation of cells on 10 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X Cell) in presence of 2 μg/ml anti-CD28 (clone 37.51, BD Biosciences). .. In vitro polarized TH1 lymphocytes were generated by adding 10 ng/ml recombinant mouse IL-12 (R&D Systems) and 20 μg/ml anti-IL-4 (clone 11B11, Bio X Cell) to the culture media during CD4+ T lymphocyte activation. .. In vitro polarized TH17 lymphocytes were generated with the addition of 20 ng/ml recombinant mouse IL-6 (R&D Systems), 20 ng/ml recombinant mouse IL-23 (R&D Systems), 4 ng/ml recombinant human (rh) TGFβ (R&D Systems) and 20 μg/ml anti-IFNγ (clone XMG1.2, Bio X Cell).
Generated:Article Title: MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.
Article Snippet: In vitro activation of CD4+ T lymphocytes was achieved by incubation of cells on 10 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X Cell) in presence of 2 μg/ml anti-CD28 (clone 37.51, BD Biosciences). .. In vitro polarized TH1 lymphocytes were generated by adding 10 ng/ml recombinant mouse IL-12 (R&D Systems) and 20 μg/ml anti-IL-4 (clone 11B11, Bio X Cell) to the culture media during CD4+ T lymphocyte activation. .. In vitro polarized TH17 lymphocytes were generated with the addition of 20 ng/ml recombinant mouse IL-6 (R&D Systems), 20 ng/ml recombinant mouse IL-23 (R&D Systems), 4 ng/ml recombinant human (rh) TGFβ (R&D Systems) and 20 μg/ml anti-IFNγ (clone XMG1.2, Bio X Cell).
Activation Assay:Article Title: MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.
Article Snippet: In vitro activation of CD4+ T lymphocytes was achieved by incubation of cells on 10 μg/ml plate-bound anti-CD3 (clone 145-2C11, Bio X Cell) in presence of 2 μg/ml anti-CD28 (clone 37.51, BD Biosciences). .. In vitro polarized TH1 lymphocytes were generated by adding 10 ng/ml recombinant mouse IL-12 (R&D Systems) and 20 μg/ml anti-IL-4 (clone 11B11, Bio X Cell) to the culture media during CD4+ T lymphocyte activation. .. In vitro polarized TH17 lymphocytes were generated with the addition of 20 ng/ml recombinant mouse IL-6 (R&D Systems), 20 ng/ml recombinant mouse IL-23 (R&D Systems), 4 ng/ml recombinant human (rh) TGFβ (R&D Systems) and 20 μg/ml anti-IFNγ (clone XMG1.2, Bio X Cell).
Incubation:Article Title: Autotaxin in encephalitogenic CD4 T cells as a therapeutic target for multiple sclerosis.
Article Snippet: T cells were plated in 48-well plates at 1 × 106 cells/well and activated with Dynabeads Mouse T-Activator CD3/CD28 beads (Gibco) at 1:10 (beads:cells). .. Recombinant mouse IL-12 (R&D Systems) and recombinant mouse IL-23 (R&D Systems) were added to the culture at 0.5 and 5 ng/mL, respectively, and cells were incubated for 48 h. Samples were collected, washed once, and replated in serum-free media for 24 h with either vehicle or HA-130 (1 μM). .. Supernatants were then collected, and an autotaxin activity assay (Eschelon Bioscience) was run.
Purification:Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity
Article Snippet: .. The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302). .. Mice C57BL/6 mice were purchased from Shanghai Model Organisms Center, Inc., and bred in our facility under specific pathogen-free conditions.
Adjuvant:Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity
Article Snippet: .. The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302). .. Mice C57BL/6 mice were purchased from Shanghai Model Organisms Center, Inc., and bred in our facility under specific pathogen-free conditions.
Immunofluorescence:Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity
Article Snippet: .. The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302). .. Mice C57BL/6 mice were purchased from Shanghai Model Organisms Center, Inc., and bred in our facility under specific pathogen-free conditions.
Cell Isolation:Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity
Article Snippet: .. The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302). .. Mice C57BL/6 mice were purchased from Shanghai Model Organisms Center, Inc., and bred in our facility under specific pathogen-free conditions.
Staining:Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity
Article Snippet: .. The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302). .. Mice C57BL/6 mice were purchased from Shanghai Model Organisms Center, Inc., and bred in our facility under specific pathogen-free conditions.
Sterility:
Article Title: Trabectedin enhances the antitumor effects of IL-12 in triple-negative breast cancer
Article Snippet: IL-12 is a potent NK cell–stimulating cytokine, but the presence of immunosuppressive myeloid cells such as myeloid-derived suppressor cells (MDSC) can inhibit IL-12–induced NK-cell cytotoxicity.. Thus, we hypothesized that trabectedin, a myeloid cell– depleting agent, would improve the efficacy of IL-12 in triplenegative breast cancer (TNBC).. In vitro treatment of healthy donor NK cells with trabectedin increased expression of the activation marker CD69 and mRNA expression of T-box transcription factor (Tbx21), the cytotoxic ligands TNF-related apoptosis–inducing ligand (TNFSF10), Fas ligand (FASLG), and the dendritic cell (DC)– recruiting chemokine lymphotactin (XCL1).
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