cd3 negative selection kit  (STEMCELL Technologies Inc)

Journal: Cells

Article Title: The Interactions of T Cells with Myeloid-Derived Suppressor Cells in Peripheral Blood Stem Cell Grafts

doi: 10.3390/cells13181545

Figure Lengend Snippet: A comparison of cellular compositions in allografts, lymphoma autografts and MM autografts. The percentages of CD34 + stem cells, CD33 + CD14 + CD15 − HLA-DR −/low M-MDSC, CD33 + CD14 − CD15 + PMN-MDSC, CD33 + CD14 − CD15 − HLA-DR − e-MDSC, CD3 + T, CD3 − CD56 + NK, CD3 + CD56 + NKT, CD3 + CD56 − TCRαβ + CD4 − CD8 − DNTreg, CD3 + CD4 + CD25 + Foxp3 + Treg, CD19 + B cells, and CD14 + HLA-DR hi conventional monocytes in the grafts were evaluated by staining the graft cells using fluorescence labeled antibodies and analyzed by Flowjo software. ( A ) Percentages of cell subsets in the grafts. ( B ) Infusion dose of cell subsets in the grafts. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CD3 + T cells were sorted from the grafts using a CD3 negative selection kit according to the manufacturer’s protocol (StemCell Technologies, Vancouver, BC, Canada).

Techniques: Comparison, Staining, Fluorescence, Labeling, Software

Journal: Cells

Article Title: The Interactions of T Cells with Myeloid-Derived Suppressor Cells in Peripheral Blood Stem Cell Grafts

doi: 10.3390/cells13181545

Figure Lengend Snippet: Characterization of T cells in the grafts. To evaluate the percentages of naïve, effector or memory T cells, fluorescence-labeled antibodies against CD45RA and CCR7 were added into the staining panel. To evaluate the proliferation and cytokine secretion of T cells in the grafts, sorted CD3 + T cells from allografts and autografts were cultured for 5 days and then were stained with Ki67 to evaluate T cell proliferation, with fluorescence-labeled anti-IFN-γ for cytokine secretion. The expression of inhibitory receptors PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and BTLA on CD4 + T and CD8 + T cells were evaluated by staining the graft cells using fluorescence-labeled antibodies. ( A ) Percentages of naïve, effector, central memory and effector memory T cells in the grafts. ( B ) The expression levels of Ki67 and IFN-γ in CD3 + T cells of 10 allografts, 9 lymphoma autografts and 10 MM autografts. ( C ) The expression pattern of inhibitory receptors on T cells in the grafts. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CD3 + T cells were sorted from the grafts using a CD3 negative selection kit according to the manufacturer’s protocol (StemCell Technologies, Vancouver, BC, Canada).

Techniques: Fluorescence, Labeling, Staining, Cell Culture, Expressing

Journal: Cells

Article Title: The Interactions of T Cells with Myeloid-Derived Suppressor Cells in Peripheral Blood Stem Cell Grafts

doi: 10.3390/cells13181545

Figure Lengend Snippet: Morphology and immune regulatory function of M-MDSCs in the allografts, lymphoma and MM autografts. M-MDSCs were sorted from allografts, lymphoma and MM autografts using flow cytometer based on cell surface markers. The morphology of M-MDSCs was evaluated by HEMA-3 staining and then read via microscopy (200× magnification). By using CD3 negative selection kit, CD3 + T cells were sorted from peripheral blood of third-party healthy donors or from PBSCs grafts of lymphoma or multiple myeloma patients. CD3 + T cells were co-cultured with M-MDSCs at different ratios in the presence of anti-CD3/CD28 for 4–5 days. T cell proliferation, IFN-γ secretion and Treg expansion were evaluated. ( A ) Morphology and immune regulatory function of M-MDSCs sorted from allograft on third-party T cell proliferation, IFN-γ secretion and CD4 + CD25 + Foxp3 + Treg expansion. ( B ) Morphology and immune regulatory function of M-MDSCs sorted from lymphoma autograft on autologous T cell proliferation, IFN-γ secretion and autologous CD4 + CD25 + Foxp3 + Treg expansion. ( C ) Morphology and immune regulatory function of M-MDSCs sorted from MM autograft on autologous T cell proliferation and IFN-γ secretion. ( D ) Comparison of suppressive function of M-MDSCs sorted from allograft or autograft against the same third-party healthy donor T cells at a ratio of T:M-MDSC (1:0.4). *, p < 0.05; **, p < 0.01; ***, p < 0.001. These results represent one of two to three independent experiments.

Article Snippet: CD3 + T cells were sorted from the grafts using a CD3 negative selection kit according to the manufacturer’s protocol (StemCell Technologies, Vancouver, BC, Canada).

Techniques: Flow Cytometry, Staining, Microscopy, Selection, Cell Culture, Comparison

Journal: Cells

Article Title: The Interactions of T Cells with Myeloid-Derived Suppressor Cells in Peripheral Blood Stem Cell Grafts

doi: 10.3390/cells13181545

Figure Lengend Snippet: M-MDSCs drive CD8 + T cell exhaustion. M-MDSCs sorted from lymphoma autograft were co-cultured with sorted autologous CD3 + T cells in the presence of anti-CD3/CD28 for 4–5 days. The T cell proliferation, expression of PD-1 and IFN-γ on CD8 + T cells and CD4 + T cells were evaluated. To block the interaction of PD-L1 with PD-1, anti-PD-L1 (10 μg/mL) was added into the co-culture system at the beginning. ( A ) The representative figures of expression of PD-1 and IFN-γ on CD8 + T cells. ( B ) Effects of M-MDSCs on autologous T cell proliferation, effector T expansion and PD-1 expression. ( C ) The effects of anti-PD-L1 on the suppressive function of M-MDSCs sorted from lymphoma autograft. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CD3 + T cells were sorted from the grafts using a CD3 negative selection kit according to the manufacturer’s protocol (StemCell Technologies, Vancouver, BC, Canada).

Techniques: Cell Culture, Expressing, Blocking Assay, Co-Culture Assay

Journal: Cells

Article Title: The Interactions of T Cells with Myeloid-Derived Suppressor Cells in Peripheral Blood Stem Cell Grafts

doi: 10.3390/cells13181545

Figure Lengend Snippet: Morphology and immune regulatory function of PMN-MDSCs in the allografts, lymphoma and MM autografts. The morphology of sorted PMN-MDSCs was evaluated by HEMA-3 staining and then read via microscopy (200× magnification). The CD3 + T cells sorted from healthy third-party donor or autologous donor were co-cultured with PMN-MDSCs at different ratios in the presence of anti-CD3/CD28 for 4–5 days. T cell proliferation, IFN-γ secretion and Treg expansion were evaluated. ( A ) Morphology and immune regulatory function of PMN-MDSCs sorted from allograft on third-party T cell proliferation, IFN-γ secretion and CD4 + CD25 + Foxp3 + Treg expansion. ( B ) Morphology and immune regulatory function of PMN-MDSCs sorted from lymphoma autograft on T cell proliferation, IFN-γ secretion and CD4 + CD25 + Foxp3 + Treg expansion. ( C ) Morphology and immune regulatory function of PMN-MDSCs sorted from MM autograft on T cell proliferation and IFN-γ secretion. ( D ) Comparison of the suppressive function of M-MDSCs and PMN-MDSCs from the same graft at a ratio of T:M-MDSC (1:0.4). These results represent one of two to three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CD3 + T cells were sorted from the grafts using a CD3 negative selection kit according to the manufacturer’s protocol (StemCell Technologies, Vancouver, BC, Canada).

Techniques: Staining, Microscopy, Cell Culture, Comparison