Journal:

Article Title: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

doi: 10.1046/j.1365-2567.1999.00728.x

Figure Lengend Snippet: FACS analysis of bone marrow cells cultured in GM-CSF. Bone marrow cells cultured for 6 days in the presence of GM-CSF were stained with mAb for MHC class II molecules for flow cytometry analysis. (a) Histogram showing two populations expressing MHC class II molecules at high (MHC-IIhi) and low levels (MHC-IIlo). (b) and (c) Histograms representing the size (FSC) and the structural features (SSC) of gated MHC-IIhi (thick line) and MHC-IIlo (thin line) cells. Few MHC class II-negative cells were observed in this experiment.

Article Snippet: Generation of DC from bone marrow Bone marrow (BM) cells were obtained from DBA/2 mice as previously described 26 and cultured at 7×10 5 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% l-glutamine, 20 μg/ml gentamicin, 50 μm 2β-mercaptoethanol, and 2000 U/ml recombinant murine (rm) GM-CSF (Genzyme, Cambridge, MA) with or without 1000 U/ml of rmIL-4 (PeproTech, London, UK) or with or without 250 ng/ml of human Flt3-L (Immunex, Seattle, WA) in 24-well plates at 37° in 5% CO 2 .

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing

Journal:

Article Title: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

doi: 10.1046/j.1365-2567.1999.00728.x

Figure Lengend Snippet: Immunophenotypical analysis of MHC-IIhi and MHC-IIlo cells cultured in GM-CSF for 6 and 9 days

Article Snippet: Generation of DC from bone marrow Bone marrow (BM) cells were obtained from DBA/2 mice as previously described 26 and cultured at 7×10 5 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% l-glutamine, 20 μg/ml gentamicin, 50 μm 2β-mercaptoethanol, and 2000 U/ml recombinant murine (rm) GM-CSF (Genzyme, Cambridge, MA) with or without 1000 U/ml of rmIL-4 (PeproTech, London, UK) or with or without 250 ng/ml of human Flt3-L (Immunex, Seattle, WA) in 24-well plates at 37° in 5% CO 2 .

Techniques: Cell Culture

Journal:

Article Title: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

doi: 10.1046/j.1365-2567.1999.00728.x

Figure Lengend Snippet: Cell surface immunophenotype of MHC class II-expressing cells obtained after 6 (a) and 9 days (b) of culture in GM-CSF alone (upper row) or in combination with Flt3-L (middle row) or IL-4(lower row). At day 6 of culture, cells were sorted by flow cytometry according to their high or low expression of MHC class II molecules. Immediately and after 3 days of culture (day 9), cells were double-stained for MHC class II molecules and for additional indicated markers. Histograms: dotted line, negative controls labelled with the same amount of isotype-matched control mAb; thick line, MHC-IIhi cells; thin line, MHC-IIlo cells. Representative data from three independent experiments performed on unsorted cells are shown.

Article Snippet: Generation of DC from bone marrow Bone marrow (BM) cells were obtained from DBA/2 mice as previously described 26 and cultured at 7×10 5 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% l-glutamine, 20 μg/ml gentamicin, 50 μm 2β-mercaptoethanol, and 2000 U/ml recombinant murine (rm) GM-CSF (Genzyme, Cambridge, MA) with or without 1000 U/ml of rmIL-4 (PeproTech, London, UK) or with or without 250 ng/ml of human Flt3-L (Immunex, Seattle, WA) in 24-well plates at 37° in 5% CO 2 .

Techniques: Expressing, Flow Cytometry, Staining

Journal:

Article Title: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

doi: 10.1046/j.1365-2567.1999.00728.x

Figure Lengend Snippet: Cell numbers in culture with GM-CSF, GM-CSF+Flt3-L, GM-CSF+IL-4 after 6 (a) and 9 (b) days. The percentages of MHC-IIhi and MHC-IIlo cells were determined by flow cytometry analysis after double-staining for MHC class II molecules and CD11c markers. These percentages allowed calculation of the number of cells obtained per well for each population. The number of cells of each population is expressed/two legs (tibias and femurs). Mean±SD of six and five independent experiments at day 6 and day 9, respectively. *Significantly different compared to GM-CSF alone.

Article Snippet: Generation of DC from bone marrow Bone marrow (BM) cells were obtained from DBA/2 mice as previously described 26 and cultured at 7×10 5 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% l-glutamine, 20 μg/ml gentamicin, 50 μm 2β-mercaptoethanol, and 2000 U/ml recombinant murine (rm) GM-CSF (Genzyme, Cambridge, MA) with or without 1000 U/ml of rmIL-4 (PeproTech, London, UK) or with or without 250 ng/ml of human Flt3-L (Immunex, Seattle, WA) in 24-well plates at 37° in 5% CO 2 .

Techniques: Flow Cytometry, Double Staining

Journal:

Article Title: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

doi: 10.1046/j.1365-2567.1999.00728.x

Figure Lengend Snippet: MLR with sorted MHC-IIhi(•) and MHC-IIlo (○) cells after a 6 (upper panel) or 9-day (lower panel) culture in GM-CSF alone (left panel) or in combination with Flt3-L (middle panel) or IL-4 (right panel). Cells from DBA/2 mice were sorted at day 6 and subcultured until day 9 as described in Fig. 2. Graded numbers of cells were irradiated and cocultured with 105 responding LN cells from C57BL/6 mice. Proliferative responses were measured (c.p.m.) on day 4, which corresponds to the peak of response under our experimental conditions.

Article Snippet: Generation of DC from bone marrow Bone marrow (BM) cells were obtained from DBA/2 mice as previously described 26 and cultured at 7×10 5 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% l-glutamine, 20 μg/ml gentamicin, 50 μm 2β-mercaptoethanol, and 2000 U/ml recombinant murine (rm) GM-CSF (Genzyme, Cambridge, MA) with or without 1000 U/ml of rmIL-4 (PeproTech, London, UK) or with or without 250 ng/ml of human Flt3-L (Immunex, Seattle, WA) in 24-well plates at 37° in 5% CO 2 .

Techniques: Irradiation

Journal:

Article Title: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

doi: 10.1046/j.1365-2567.1999.00728.x

Figure Lengend Snippet: FACS analysis of popliteal LN cells 24 hr after injection of cells cultured in GM-CSF, GM-CSF+Flt3-L, or GM-CSF+IL-4 for 6(upper panel) or 9 days (lower panel). Cells were stained with PKH2 and injected s.c into the footpad of syngeneic mice. Twenty-four hours later, the popliteal draining LN were harvested and digested by collagenase treatment. LN cells (1·5×105) were analysed by flow cytometry for their size (forward scatter) and PKH2 green fluorescence. Controls were popliteal LN cells from non-injected mice. Representative data from one of three independent experiments performed with three or four mice per group.

Article Snippet: Generation of DC from bone marrow Bone marrow (BM) cells were obtained from DBA/2 mice as previously described 26 and cultured at 7×10 5 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% l-glutamine, 20 μg/ml gentamicin, 50 μm 2β-mercaptoethanol, and 2000 U/ml recombinant murine (rm) GM-CSF (Genzyme, Cambridge, MA) with or without 1000 U/ml of rmIL-4 (PeproTech, London, UK) or with or without 250 ng/ml of human Flt3-L (Immunex, Seattle, WA) in 24-well plates at 37° in 5% CO 2 .

Techniques: Injection, Cell Culture, Staining, Flow Cytometry, Fluorescence

Journal:

Article Title: Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

doi: 10.1046/j.1365-2567.1999.00728.x

Figure Lengend Snippet: Effect of vaccination with MHC-IIhi and MHC-IIlo cell populations generated in GM-CSF culture pulsed with hCD4 soluble antigen. Survival of mice vaccinated with sorted hCD4-pulsed MHC-IIhi and MHC-IIlo cell populations, unsorted, pulsed or unpulsed BM-derived cells, after challenge with L1210/hCD4 tumour cells (five mice/group).

Article Snippet: Generation of DC from bone marrow Bone marrow (BM) cells were obtained from DBA/2 mice as previously described 26 and cultured at 7×10 5 cells/ml in RPMI-1640 supplemented with 5% FCS, 1% l-glutamine, 20 μg/ml gentamicin, 50 μm 2β-mercaptoethanol, and 2000 U/ml recombinant murine (rm) GM-CSF (Genzyme, Cambridge, MA) with or without 1000 U/ml of rmIL-4 (PeproTech, London, UK) or with or without 250 ng/ml of human Flt3-L (Immunex, Seattle, WA) in 24-well plates at 37° in 5% CO 2 .

Techniques: Generated, Derivative Assay