Untreated or XBP1/TRP2hsp70 or control DNA-transfected DC (6×104) were co-cultured with syngeneic CD8+T cells (3×105) isolated from splenocytes of naïve B6 mice using anti-mouse CD8 microbeads (Miltenyi Biotec) in 200 μl RPMI 1640 containing 10% (v/v) FBS for 3 days. 3H-thymidine (1 μCi/well; Du Pont/New England Nuclear, Boston, MA) was added during the last 16–18 hours of culture. 3H-thymidine incorporation was then measured using a scintillation counter (Packard, Meriden, CT) (23 (link)). In another set of experiments, untreated or XBP1/NeuEDhsp70 or control DNA-transfected DC (2x104) were co-cultured with syngeneic naïve splenic T cells (1x105) purified from naïve BALB/c mice using a mouse T cell isolation kit (R&D Systems) in 200 μl RPMI1640 media containing 10% (v/v) FBS for 5 days. IFN-γ in the culture supernatants were determined by ELISA.
Anti mouse cd8 microbeads
Manufactured by Miltenyi Biotec
Anti-mouse CD8 microbeads are magnetic beads coated with antibodies specific for the mouse CD8 surface marker. They are used for the isolation of CD8+ T cells from mouse tissue samples.
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Lab products found in correlation
3h thymidine, by Hewlett-Packard (1 mentions)
Mouse t cell isolation kit, by R&D Systems (1 mentions)
Scintillation counter, by Hewlett-Packard (1 mentions)
3h thymidine, by DuPont (1 mentions)
Midimacs magnet, by Miltenyi Biotec (1 mentions)
Cd4 cd62l isolation kit, by Miltenyi Biotec (1 mentions)
Facsaria 1, by BD (1 mentions)
Anti mouse dx5 microbeads, by Miltenyi Biotec (1 mentions)
Balb c mice, by Charles River Laboratories (1 mentions)
3 protocols using anti mouse cd8 microbeads
1
T Cell Activation Assay with Dendritic Cells
2
Isolation of Lymphocyte Subsets from Mouse Spleen
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Different lymphocyte subsets were purified from splenic mononuclear cells isolated from Balb/c mice (Charles River Laboratories, Wilmington, MA, USA). If necessary, further isolation was performed by magnetic activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) or by FACS (FACSAria I, BD Biosciences, San Jose, USA).
Briefly, cell suspension of the spleen was prepared by cutting small pieces and gently pressing through a 100 μm wire mesh. DX5+ cells were purified using anti-mouse-DX5+ MicroBeads (Miltenyi Biotec). Cells were passed through a MACS column (type LS) attached to a MidiMACS magnet (Miltenyi Biotec). DX5+ cells were collected in the positive fraction. DX5+ splenocytes were labeled with FITC-conjugated anti-mouse CD3 molecular complex (clone: 17A2, rat IgG2b) and PE-conjugated anti-mouse CD49b (clone: DX5, rat IgM) (all from BD Biosciences) for further DX5+NKT cell isolation by FACS sorting. CD4+CD62Lhigh and CD4+CD62Llow cells were purified using the CD4+CD62L+ Isolation Kit (Miltenyi Biotec) and CD8+ cells by using anti-mouse-CD8+ MicroBeads (Miltenyi Biotec).
Briefly, cell suspension of the spleen was prepared by cutting small pieces and gently pressing through a 100 μm wire mesh. DX5+ cells were purified using anti-mouse-DX5+ MicroBeads (Miltenyi Biotec). Cells were passed through a MACS column (type LS) attached to a MidiMACS magnet (Miltenyi Biotec). DX5+ cells were collected in the positive fraction. DX5+ splenocytes were labeled with FITC-conjugated anti-mouse CD3 molecular complex (clone: 17A2, rat IgG2b) and PE-conjugated anti-mouse CD49b (clone: DX5, rat IgM) (all from BD Biosciences) for further DX5+NKT cell isolation by FACS sorting. CD4+CD62Lhigh and CD4+CD62Llow cells were purified using the CD4+CD62L+ Isolation Kit (Miltenyi Biotec) and CD8+ cells by using anti-mouse-CD8+ MicroBeads (Miltenyi Biotec).
3
Adoptive Transfer of Tumor-Specific CD8+ T Cells
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Splenocytes or mesentery lymph node cells were isolated 9 or 20 d post tumor cell injection from different groups of the treated mice and stained with APC-anti-mouse CD8+ Ab to determine CD8+ T cell population (post Fc receptor blockade by α-CD16/32 Ab) by flow cytometry. CD8+ T cells (4 × 105) were purified from splenocytes of different groups of treated or naive BALB/c mice using anti-mouse CD8+ microbeads following vendor's protocols (Miltenyi Biotec, San Diego, CA) and restimulated with mitomycin C-treated AB12-luc cells (4 × 104) or CA51 colon cells (tumor-specific control; 4 × 104) in the presence of 4,000-rad-irradiated naive CD8+-depleted BALB/c splenocytes (2 × 106) in 200 μL RPMI 1640 medium supplemented with 10% FBS at 37°C, 5% CO2 for 3 d The concentration of IFNγ in the culture supernatants was tested using ELISA kit according to the manufacturer's instructions (BioLegend).
As for adoptive cell transfer, single splenocytes were also isolated from tumor-bearing BALB/c mice treated with vvDD-CXCL11, which survived more than 60 d, and adoptively transferred into naive BALB/c mice by tail vein injection at 3.0 × 107 cells per mouse. The age-matched naive BALB/c mice received PBS injection as a control. The next day, these mice were challenged with 4 × 105 AB12-luc cancer cells intraperitoneally.
As for adoptive cell transfer, single splenocytes were also isolated from tumor-bearing BALB/c mice treated with vvDD-CXCL11, which survived more than 60 d, and adoptively transferred into naive BALB/c mice by tail vein injection at 3.0 × 107 cells per mouse. The age-matched naive BALB/c mice received PBS injection as a control. The next day, these mice were challenged with 4 × 105 AB12-luc cancer cells intraperitoneally.
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