anti ccr7 fitc mab  (R&D Systems)

Journal: PLoS ONE

Article Title: IRX-2, a Novel Immunotherapeutic, Enhances Functions of Human Dendritic Cells

doi: 10.1371/journal.pone.0047234

Figure Lengend Snippet: ( A ) DC obtained from HNSCC patients were matured for 48 h either with IRX-2 or the conventional maturation cocktail. While conventionally matured DC (white bars) expressed higher levels of CD80, CD83 and CD86 (*, p<0.05), IRX-2 matured DC (black bars) showed higher expression of CD11c, CD40 and CCR7 (*, p<0.05). The data are mean x-fold of MFI ± SEM for cells obtained from 12 different HNSCC patients. ( B ) Representative histograms showing expression of DC markers after maturation with IRX-2 or the conventional cytokine cocktail in DC generated from monocytes of one HNSCC patient. The shaded peaks represent isotype controls. ( C ) Migration of mDC in vitro : Migration assays were performed as described in Materials & Methods using DC generated from peripheral blood monocytes of HNSCC patients. While iDC showed very little migration in response to CCL21, both conventional- and IRX-2-matured DC migrated significantly better. Results are shown as the mean absolute numbers of migrated cells ± SEM obtained from 5 different HNSCC patients.

Article Snippet: Anti-CCR7-FITC mAb was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Generated, Migration, In Vitro

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Immunoregulatory effects of Lurbinectedin in chronic lymphocytic leukemia

doi: 10.1007/s00262-020-02513-y

Figure Lengend Snippet: B cells are sensitive to Lurbinectedin. a CLL PBMC were incubated with Lurbinectedin 1, 3, 10, 30 nM or vehicle for 24, 48 and 72 h. Cells were stained with anti-CD19-PECy5 mAb and Annexin V-FITC. Shown are the individual values and the mean ± SEM from 10 samples. b The experiment on (a) was repeated using HD PBMC. Shown are the individual values and the mean ± SEM from 13 samples after 24 h of treatment. Statistical analysis was performed using Friedman test followed by the Dunn’s multiple comparison post-test. c, d B-CLL cells were treated with Lurbinectedin 10 nM for 24 h. c Then, γ-H2AX and PARP fragmentation were evaluated by western blot. d Bands on the immunoblots were quantified. Results are shown as the mean ± SEM of the ratio γ-H2AX/tubulin and PARP/tubulin. Statistical analysis was performed using Wilcoxon’s matched-pairs signed-rank test. Lur Lurbinectedin; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Article Snippet: Annexin-V-FITC, PE, FITC or PECy5-conjugated mAbs anti-CCR7 (clone 3D12), anti-CXCR4 (clone 12G5), anti-CD56 (clone B159), anti-HLA-DR (clone G46-6), anti-CD3 (HIT3a), anti-CD4 (OKT4), anti-CD8 (HIT8a), anti-phosphorylated Akt (clone M89-61), anti-cleaved PARP (clone F21-852), control Abs with irrelevant specificities (isotype control) and human IL-1β ELISA Set II, were obtained from BD Biosciences (USA).

Techniques: Incubation, Staining, Comparison, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Immunoregulatory effects of Lurbinectedin in chronic lymphocytic leukemia

doi: 10.1007/s00262-020-02513-y

Figure Lengend Snippet: Effects of Lurbinectedin on tumor-microenvironment cells. a Monocytes were treated with different concentrations of Lurbinectedin for 24 h. Cell death was determined by Annexin V-FITC staining and flow cytometry analysis. The individual values and the mean ± SEM are shown, n = 6. b The sensitivity of Mo-MDSC to Lurbinectedin was determined by incubating PBMC with or without Lurbinectedin 1 nM for 24 h. Cells were stained with anti-CD3 PECy5, anti-CD19 PECy5, anti-CD56 PeCy5, anti-CD14-PE and anti-HLA-DR-FITC antibodies. Mo-MDSC population was selected as Lin− (CD3−, CD19−, CD56−), CD14+ and HLA-DRlow. c NLC were incubated with vehicle, Lurbinectedin 10 or 30 nM for 24 h. Then, cells were labeled with Annexin V-FITC and analyzed by flow cytometry. The individual values and the mean ± SEM are shown, n = 6. d CLL PBMC were plated over immobilized anti-CD3 antibody (anti-CD3i) or isotype control. After 48 h of incubation, Lurbinectedin or vehicle were added for another 72 h of culture. Finally, cells were labeled with anti-CD4-PECy5 and anti-CD8-PE antibodies and analyzed by flow cytometry. The percentage of total non-viable T lymphocytes after 5 days of culture is shown (mean ± SEM), n = 11. Statistical significance was determined by the Friedman test followed by Dunn’s post-test of multiple comparisons (a, c and d) or Wilcoxon signed-rank test (b). Lur Lurbinectedin, N.S no statistically significant difference, *p < 0.05; **p < 0.01; ****p < 0.0001

Article Snippet: Annexin-V-FITC, PE, FITC or PECy5-conjugated mAbs anti-CCR7 (clone 3D12), anti-CXCR4 (clone 12G5), anti-CD56 (clone B159), anti-HLA-DR (clone G46-6), anti-CD3 (HIT3a), anti-CD4 (OKT4), anti-CD8 (HIT8a), anti-phosphorylated Akt (clone M89-61), anti-cleaved PARP (clone F21-852), control Abs with irrelevant specificities (isotype control) and human IL-1β ELISA Set II, were obtained from BD Biosciences (USA).

Techniques: Staining, Flow Cytometry, Incubation, Labeling

Journal: Journal of Periodontal & Implant Science

Article Title: Targeting the epitope spreader Pep19 by naïve human CD45RA + regulatory T cells dictates a distinct suppressive T cell fate in a novel form of immunotherapy

doi: 10.5051/jpis.2017.47.5.292

Figure Lengend Snippet: Fluorescence-activated cell sorting profiles of CD95-mediated apoptosis in expanded Tregs of a representative subject after 24 hours of incubation. While 127-Tregs and Pep19-127-Tregs demonstrated a similar resistance to CD95L-mediated apoptosis, 45RA-Tregs and Pep19-45RA-Tregs exhibited slightly greater resistance. Treg: regulatory T cell, 127-Tregs: CD4 + CD25 + CD127 lo− regulatory T cells, Pep19: peptide 19, 45RA-Tregs: CD4 + CD25 + CD45RA + regulatory T cells, PI: propidium iodide, PE: phycoerythrin, FITC: fluorescein isothiocyanate.

Article Snippet: Each set of expanded Tregs was examined for the expression of CD62L and CCR7 by staining with a PE-Cy5-labeled anti-CD4 mAb, an FITC-labeled anti-CCR7 mAb (BD Bioscience), and a PE-labeled anti-CD62L mAb (eBioscience) on the BD FACSCalibur instrument after staining with mAb cocktails as described above.

Techniques: Fluorescence, FACS, Incubation