Journal: American journal of clinical and experimental immunology

Article Title: An integrated nano-scale approach to profile miRNAs in limited clinical samples

doi:

Figure Lengend Snippet: Isolation of T cells from bronchial biopsy specimens. A. Human bronchial biopsy specimens were enzymatically dispersed and cells stained with fluorescently conjugated antibodies (Lin1 cocktail (includes CD3, CD14, CD16, CD19, CD20, and CD56) FITC-conjugated, anti-EpCAM PerCP-Cy5.5-conjugated, anti-CD8 APC-conjugated, anti-CD3 PE-Cy7-conjugated, anti-HLA-DR APC-Cy7-conjugated) and propidium iodide for analysis on the FACSAriaTM. FACS plots illustrate the gating strategy used to identify T cells in these bronchial biopsy specimens. Utilizing this gating strategy, T cells populations were sorted by FACS. The FACS plots of the post-sort samples are also displayed. B. The mean and range of the numbers of T cells obtained from bronchial biopsy specimens and BAL of healthy controls (HC), mild (MA) and moderate (MO) asthmatic patients are displayed. C. From 100ml of blood, using the memory CD4+ T cell isolation kit, PBMC-CD4+ memory cell were first selected and were subsequently stained with fluorescently conjugated antibodies (anti-CD3 PE-Cy7-conjugated, anti-CD45RA FITC-conjugated, anti-CD4 APC-Cy7-conjugated, anti-CCR4 PE-conjugated, and anti-CD25 APC-conjugated) and sorted on the FACSAriaTM to obtain two cell populations: CD4+CD45RA-CCR4- and CD4+CD45RA-CCR4+CD25-. Naïve T cells were sorted from the non-CD4+ memory cells following staining with anti-CD3 PE-Cy7-conjugated, anti-CD45RA FITC-conjugated, anti-CD4 PerCP-Cy-5.5-conjugated, anti-CD62L APC-Cy7-conjugated and anti-CD45RO PE-conjugated antibodies. The FACS plots illustrating the gating strategy and the post-sort cell populations are displayed.

Article Snippet: Cells were then stained with fluorescently conjugated antibodies (Lin1 cocktail (includes CD3, CD14, CD16, CD19, CD20, and CD56) FITC-conjugated, anti-EpCAM PerCP-Cy5.5-conjugated, anti-CD8 APC-conjugated, anti-CD3 PE-Cy7-conjugated, anti-HLA-DR APC-Cy7-conjugated, all from BD Biosciences, oxford, UK) and propidium iodide prior to analysis and sorting on the FACSAria TM (BD Biosciences, oxford, UK).

Techniques: Isolation, Staining, Cell Isolation

Journal: American journal of clinical and experimental immunology

Article Title: An integrated nano-scale approach to profile miRNAs in limited clinical samples

doi:

Figure Lengend Snippet: Airway T cells have a distinct miRNA signature compared to circulating T cells. A. Using nano-scale PCR, miRNA expression levels were measured in CD3+ T cells isolated from matched bronchial biopsy, BAL and blood specimens of 3 healthy controls, 6 mild and 6 moderate asthmatic patients. Expression levels of miRNAs (ΔCt) in each sample were first calculated by subtracting the Ct value of the miRNA from the mean Ct value of the housekeeping miRNAs, miR-103 and miR-191, in that sample. The dot-plot displays the correlation in miRNA expression levels (ΔCt) between T cells obtained from bronchial biopsy and blood specimens, across the study groups. B. The graph displays the difference in the expression levels (ΔΔCt) between blood and bronchial biopsy T cells (ΔΔCt= ΔCt blood - ΔCt bronchial biopsy). All miRNAs indicated on the figure were differentially expressed in airway T cells compared to blood T cells i.e. airway-specific are shown. miR-125b, miR-19a, miR-19b and miR-106b had expression values (ΔΔCt) significantly different between healthy subjects and asthmatic patients. p-values were obtained by ANOVA, * p <0.01. C. Dot-plots display the correlation in miRNA expression levels between T cells obtained from bronchial biopsy and BAL specimens, across the study groups. D. A panel of 96 miRNAs (mix 2) was measured in BAL and sputum supernatant samples by using the nano-scale PCR method. The venn diagram illustrates the overlap in the miRNAs detected in the two samples. E. The dot-plot displays the correlation in miRNA expression levels (Ct values) between BAL and sputum supernatant samples.

Article Snippet: Cells were then stained with fluorescently conjugated antibodies (Lin1 cocktail (includes CD3, CD14, CD16, CD19, CD20, and CD56) FITC-conjugated, anti-EpCAM PerCP-Cy5.5-conjugated, anti-CD8 APC-conjugated, anti-CD3 PE-Cy7-conjugated, anti-HLA-DR APC-Cy7-conjugated, all from BD Biosciences, oxford, UK) and propidium iodide prior to analysis and sorting on the FACSAria TM (BD Biosciences, oxford, UK).

Techniques: Expressing, Isolation

Journal: Journal of Translational Medicine

Article Title: First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity

doi: 10.1186/1479-5876-8-71

Figure Lengend Snippet: IMP321 increases the numbers of monocytes, NK and activated CD8 T cells in blood (panel A) . Fresh blood samples were collected pre-dosing, at D1, D85 and D170 and directly stained with fluorochrome-conjugated antibodies in tubes containing a precise number of fluorescent control beads. The results show the mean ± sd of the absolute numbers of CD45 + CD14 + (monocytes), CD3 - CD56 + (NK cells) and CD38 + HLA-DR + CD8 + (activated CD8 + cells) cells. The paired non-parametric Wilcoxon signed rank test was used to compare increases observed between D85 or D170 and D1. When significant (< 0.05), p values are indicated. IMP321 increases the percentages of dendritic cells and cytotoxic CD45RA + Effector-Memory CD8 + T cells (EMRA) in PBMCs (panel B). PBMCs cells collected pre-dosing, at D1, D85 and D170 were isolated and stained with fluorochrome-conjugated antibodies and analyzed by flow cytometry. The results show the mean ± sd of the percentages of plasmacytoid dendritic cells (pDC, CD45 + CD14 - CD16 - HLA-DR + CD123 + CD11c - ) and myeloid dendritic cells (mDC, CD45 + CD14 - CD16 - HLA-DR + CD123 - CD11c + ) in CD45 + cells, as well as the percentages of CD45RA + CD45RO - CD62L - in the CD8 + T cell population (CD45RA + EM CD8 T cells or EMRA). The significant Wilcoxon p values are indicated.

Article Snippet: Blood samples were collected pre-dosing at D1, D85 and D170 and directly stained with BD Multitest CD8-FITC/CD38-PE/CD3-PerCP/HLA-DR-APC, with BD Multitest 6-color TBNK (CD3-FITC/CD16-PE+CD56-PE/CD45-PerCPCy5.5/CD4PE-Cy7/CD19-APC/CD8-APC-Cy7), BD Simultest LeucoGate (CD45-FITC/CD14-PE) in tubes containing a precise number of fluorescent control beads (BD Trucount™tubes, BD Biosciences).

Techniques: Staining, Isolation, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity

doi: 10.1186/1479-5876-8-71

Figure Lengend Snippet: IMP321 increases the expression of activation markers on blood monocytes . Blood samples were collected pre-dosing, at D1, D85 and D170 and directly stained with fluorochrome-conjugated CD45, CD14, anti-HLA-DR and CD11a, CD11b, CD16, CD35, CD54, CD64, CD80 or CD86 antibodies in tubes containing a precise number of fluorescent control beads. The expression of activation markers on monocytes was directly proportional to the cell-bound fluorescence. The results shown in panel A are the mean ± sd after normalization of the cell-bound fluorescence against the fluorescence of control beads. Statistically significant increases between D85 or D170 and D1 are analyzed using Wilcoxon signed rank test and significant p values (< 0.05) are shown. In panel B, the percentage of patients showing increases in the expression of the indicated numbers of activation markers at D85 or D170 compared to the baseline at D1 was calculated. The number of markers (n) displaying an increase by at least 50% was calculated for each patient in the 1.25 mg (7 patients) and 6.25 mg (12 patients) groups. The pie charts represent the percentages of patients with increases in the indicated number of markers.

Article Snippet: Blood samples were collected pre-dosing at D1, D85 and D170 and directly stained with BD Multitest CD8-FITC/CD38-PE/CD3-PerCP/HLA-DR-APC, with BD Multitest 6-color TBNK (CD3-FITC/CD16-PE+CD56-PE/CD45-PerCPCy5.5/CD4PE-Cy7/CD19-APC/CD8-APC-Cy7), BD Simultest LeucoGate (CD45-FITC/CD14-PE) in tubes containing a precise number of fluorescent control beads (BD Trucount™tubes, BD Biosciences).

Techniques: Expressing, Activation Assay, Staining, Fluorescence

Journal: Gastroenterology

Article Title: ACTIVATION OF NATURAL KILLER CELLS DURING ACUTE INFECTION WITH HEPATITIS C VIRUS

doi: 10.1053/j.gastro.2010.01.006

Figure Lengend Snippet: Panel A: representative flow cytometry dot plot of the CD56bright CD3− and CD56dim CD3− populations in acute HCV infection. Panel B: frequency of NK cells as a percentage of the lymphocyte population and the frequencies of CD56bright and CD56dim NK cells as a percentage of the total NK population in 14 chronically evolving patients (CH, circles), 8 self-limited patients (SL, squares) and 17 healthy controls (CTR, triangles). Panel C: changes in NK cells in the follow-up phase of infection. The upper panel shows NK cells as a percentage of the live lymphocyte gate, and the middle and lower panels show the relative proportions of CD56bright (middle) and CD56dim (lower) NK cells.

Article Snippet: PBMC were incubated with the following Abs: CD56-FITC (BD Biosciences-Pharmingen, San Jose, CA); NKG2D-PE (R&D Systems, Inc., Minneapolis, MN), and CD3-PerCP, CD158b-FITC (KIR2DL2/2DL3/2DS2), CD56-PE-Cy7, CD3-APC-Cy7 (BD Biosciences-Pharmingen), 3DL1-biotin and Streptavidin-PerCP (Abcam, Cambridge, UK), CD158a-APC (KIR2DL1/2DS1; Beckman Coulter, High Wycombe, UK) and analysed on a BD Biosciences flow cytometer (FACSCalibur or FACSCanto).

Techniques: Flow Cytometry, Infection

Journal: Gastroenterology

Article Title: ACTIVATION OF NATURAL KILLER CELLS DURING ACUTE INFECTION WITH HEPATITIS C VIRUS

doi: 10.1053/j.gastro.2010.01.006

Figure Lengend Snippet: PBMCs were incubated overnight with or without IL-12. The cells were then stained with mAbs to detect IFN-γ and identify NK cells (CD56+ CD3−). Panel A: representative flow cytometry dot plots of IFN-γ producing cells following overnight culture with or without IL-12. Panel B: percentage of IFN-γ+ total NK (upper plot) and IFN-γ+ CD56bright NK cells (lower plot) in IL-12 stimulated PBMC in the various patient populations in the acute phase of infection (8 self-limited, 14 chronically evolving, 17 controls). Panel C: IFN-γ production during the follow-up phase of HCV infection in individuals with self-limited and chronic hepatitis on total NK cells and on CD56bright NK cells.

Article Snippet: PBMC were incubated with the following Abs: CD56-FITC (BD Biosciences-Pharmingen, San Jose, CA); NKG2D-PE (R&D Systems, Inc., Minneapolis, MN), and CD3-PerCP, CD158b-FITC (KIR2DL2/2DL3/2DS2), CD56-PE-Cy7, CD3-APC-Cy7 (BD Biosciences-Pharmingen), 3DL1-biotin and Streptavidin-PerCP (Abcam, Cambridge, UK), CD158a-APC (KIR2DL1/2DS1; Beckman Coulter, High Wycombe, UK) and analysed on a BD Biosciences flow cytometer (FACSCalibur or FACSCanto).

Techniques: Incubation, Staining, Flow Cytometry, Infection

Journal: Gastroenterology

Article Title: ACTIVATION OF NATURAL KILLER CELLS DURING ACUTE INFECTION WITH HEPATITIS C VIRUS

doi: 10.1053/j.gastro.2010.01.006

Figure Lengend Snippet: Panel A: representative flow cytometry dot plot to illustrate the gating strategy used to obtain NK cells single positive (SP) for one specific KIR group, in this case KIR2DL1/S1. In this plot CD3− CD56dim NK cells were gated and NK cells negative for both KIR2DL2/L3/S2 and KIR3DL1 were analysed for expression of KIR2DL1/S1. The histogram shows IFN-γ production by these KIR2DL1/S1 positive NK cells. Panel B: percentage of IFN-γ producing NK cells single positive for KIR2DL1/S1 or KIR2DL2/3/S2 in the acute phase of infection in patients with acute hepatitis and healthy controls. Circles depict patients with chronically evolving infection (n=12), squares individuals with self-limited infection (n=7) and triangles represent the controls (n=17).

Article Snippet: PBMC were incubated with the following Abs: CD56-FITC (BD Biosciences-Pharmingen, San Jose, CA); NKG2D-PE (R&D Systems, Inc., Minneapolis, MN), and CD3-PerCP, CD158b-FITC (KIR2DL2/2DL3/2DS2), CD56-PE-Cy7, CD3-APC-Cy7 (BD Biosciences-Pharmingen), 3DL1-biotin and Streptavidin-PerCP (Abcam, Cambridge, UK), CD158a-APC (KIR2DL1/2DS1; Beckman Coulter, High Wycombe, UK) and analysed on a BD Biosciences flow cytometer (FACSCalibur or FACSCanto).

Techniques: Flow Cytometry, Expressing, Infection