Journal:

Article Title: Toll-Like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

doi: 10.1016/j.immuni.2006.04.008

Figure Lengend Snippet: TLR stimulation allows lymphoid biased progenitors to produce dendritic cells at the expense of B lymphopoiesis.(A) Sorted CLPs (5,000/well) from C57BL/6 or Myd88-/- mice were stimulated in X-VIVO15 medium alone, LPS (10 μg/ml) or Pam3CSK4 (100 ng/ml) plus SCF, FL and IL-7. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graphs depict cell yields for CD19+ cells or Mac-1+ cells (Right) and the data represent mean values with standard deviations from triplicate cultures (*P<0.02). The results are representative of those obtained in five independent experiments. (B) Subsets of the recovered cells described in panel (A) were sorted and used to prepare Giemsa-May-Grünwal stained slides. (C) Cultured cells from C57BL/6 mice were also analyzed by flow cytometry for expression of Gr-1 and CD11c. (D) Sorted CLPs from C57BL/6 mice were cultured in the presence of SCF, FL and IL-7 with a range of concentrations of LPS or combinations of recombinant mouse CD14-Fc protein (1 μg/ml) plus LPS. After 7 days in culture, cells were analyzed by flow cytometry for expression of CD19 and Mac-1 (Left). The bar graph depicts cell yields for CD19+ cells or Mac-1+ cells (Right). Data represent mean values with standard deviations from triplicate cultures (*P<0.003). The results are representative of three independent experiments.

Article Snippet: X-VIVO15 medium, that seemed to be optimal for lymphoid cultures, contained 1% detoxified bovine serum albumin (Stem Cell Technologies, Vancouver, Canada), 5×10 -5 M 2-mercaptoethanol (2-ME), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin.

Techniques: Flow Cytometry, Expressing, Staining, Cell Culture, Recombinant

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor

doi: 10.1007/s00262-014-1572-5

Figure Lengend Snippet: Expansion of patient-derived T cells after elutriation. Leukaphereses from melanoma patients were elutriated, and cells from fraction 3 were expanded in presence of the anti-CD3 antibody OKT3 and IL-2 for 10 days. a Cells were expanded directly after elutriation in the following cell culture media: X-vivo 15 (n = 4), OpTmizer (n = 4), DC-medium (n = 3). b Cells were left resting for 1 day after elutriation and expanded in X-vivo 15 medium supplemented with either OKT3 antibody and IL-2 (n = 3) or additional IL-7 and IL-15 (n = 3). The different symbols indicate individual patients, and the mean fold expansion is depicted as a bar

Article Snippet: T cells (5 × 10 4 ) were co-cultured with the same number of tumor cells in 100 µl X-vivo 15 medium for 16 h. The cytokine concentrations in the supernatants were determined using the human Th 1 /Th 2 Cytokine Kit II (BD Bioscience, Franklin Lakes, NJ, USA) according to manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor

doi: 10.1007/s00262-014-1572-5

Figure Lengend Snippet: Scheme of the preferred protocol. PBMC are seeded at a concentration of 106/ml in X-vivo 15 supplemented with 103 IU/ml Proleukin (IL-2) and 0.1 µg/ml anti-CD3 antibody (OKT-3). Fresh IL-2 (103 IU/ml) is added at the indicated time points (103 IU/ml final volume). On day 3, the cells are counted and diluted to 2 × 105 cells/ml in X-vivo 15. The volume is doubled with X-vivo 15 on day 7. Cells are electroporated on day 10 and cryoconserved 2 h after electroporation

Article Snippet: T cells (5 × 10 4 ) were co-cultured with the same number of tumor cells in 100 µl X-vivo 15 medium for 16 h. The cytokine concentrations in the supernatants were determined using the human Th 1 /Th 2 Cytokine Kit II (BD Bioscience, Franklin Lakes, NJ, USA) according to manufacturer’s instructions.

Techniques: Concentration Assay, Electroporation