mouse anti human panck igg1  (Bio-Rad)

Journal: Cancers

Article Title: High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients

doi: 10.3390/cancers12010127

Figure Lengend Snippet: Optimization of Labyrinth for cell recovery. ( A ) The Labyrinth was loaded with red dye to show the device’s structure as well as the inlet and outlets. ( B , C ) Using the H1650 lung cancer cell line, different flow rates ranging from 2300–2500 μL/min were tested for inertial separation of cancer cells. Pre-labeled H1650 cell line and DAPI (4′,6-diamidino-2-phenylindole)-labeled white blood cells (WBCs) (1000 cells) were spiked into PBS and processed through the Labyrinth. Using a flow rate of 2500 μL/min, 82% ± 5% of H1650 cells were recovered from outlet #2 and 78% ± 18% of WBCs were removed through outlet #1. ( D ) Immunofluorescence staining optimization. Anti-human CD45 (cluster of differentiation 45) (green), anti-human PanCK (pan-Cytokeratin) (red), anti-human EpCAM (Epithelial cell adhesion molecule) (orange), and anti-human Vimentin (pink) antibodies were tested with lung cancer cell lines, H1975 and A549.

Article Snippet: The panel of antibodies (mouse anti-human CD45 (IgG2a, Biorad, Hercules, CA, USA), mouse anti-human PanCK (IgG1 Biorad, Hercules, CA, USA), anti-human EpCAM, biotinylated (goat IgG, R&D systems, Minneapolis, MN, USA), and rabbit anti-human Vimentin (Abcam, Cambridge, MA, USA) were optimized using lung cancer cell lines including H1975 and A549.

Techniques: Cell Recovery, Labeling, Immunofluorescence, Staining

Journal: Cancers

Article Title: High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients

doi: 10.3390/cancers12010127

Figure Lengend Snippet: Isolation of circulating tumor cells (CTCs) from non-small-cell lung cancer (NSCLC) patients ( n = 25). ( A ) Fluorescent microscope image of a single CTC. Cells are stained with DAPI (blue), PanCK (red) and CD45 (green). ( B , C ) Confocal microscopy images of some CTC clusters. ( D ) An individual bar plot of the number of CTCs recovered from NSCLC patient samples at baseline, using Labyrinth. The overall number of CTCs in NSCLC patient samples was 417 ± 1023 per mL, while healthy controls had 1 ± 1.7 CTCs per mL.

Article Snippet: The panel of antibodies (mouse anti-human CD45 (IgG2a, Biorad, Hercules, CA, USA), mouse anti-human PanCK (IgG1 Biorad, Hercules, CA, USA), anti-human EpCAM, biotinylated (goat IgG, R&D systems, Minneapolis, MN, USA), and rabbit anti-human Vimentin (Abcam, Cambridge, MA, USA) were optimized using lung cancer cell lines including H1975 and A549.

Techniques: Isolation, Microscopy, Staining, Confocal Microscopy

Journal: Cancers

Article Title: High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients

doi: 10.3390/cancers12010127

Figure Lengend Snippet: Identification of heterogeneous CTC subpopulations isolated from NSCLC patient samples ( n = 23). ( A ) Fluorescent microscope images of different subpopulations of CTCs (CTCs, EpCAM+ CTCs, Vimentin+ CTCs, and Double+ CTCs). Cells are stained with DAPI (blue), CD45 (green), PanCK (red), EpCAM (orange), and Vimentin (pink). ( B , C ) The percentage of CTCs expressing both EpCAM ( B ) and Vimentin ( C ) recovered from each NSCLC patient sample ( n = 23). The EpCAM+/− CTCs is shown in dark/light orange respectively and the Vimentin+/− CTCs is shown in dark/light pink respectively. An average of 31% of the captured CTCs were EpCAM+ and 69% were EpCAM− CTCs. An average of 45% of the captured CTCs were Vimenin+ and 55% were Vimentin− CTCs. The total number of CTCs/mL across all patient samples is shown on the bottom of the graph.

Article Snippet: The panel of antibodies (mouse anti-human CD45 (IgG2a, Biorad, Hercules, CA, USA), mouse anti-human PanCK (IgG1 Biorad, Hercules, CA, USA), anti-human EpCAM, biotinylated (goat IgG, R&D systems, Minneapolis, MN, USA), and rabbit anti-human Vimentin (Abcam, Cambridge, MA, USA) were optimized using lung cancer cell lines including H1975 and A549.

Techniques: Isolation, Microscopy, Staining, Expressing

Journal: Cancers

Article Title: High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients

doi: 10.3390/cancers12010127

Figure Lengend Snippet: Isolation of CTC clusters from NSCLC patient samples ( n = 25) by using Labyrinth. ( A ) Immunofluorescence staining images of the representative recovered CTC clusters in NCSLC patients. CTC clusters are stained with DAPI (blue), CD45 (green), PanCK (red), EpCAM (orange), and Vimentin (pink). ( B ) The percentage of CTCs in single (light pink) vs. cluster (red) forms. Across all patients, only one patient did not have CTC clusters. ( C ) Comparison between the total CTC numbers in single and cluster forms. Significantly higher numbers of clusters compared to the single CTCs were observed in the captured CTCs from NSCLC patients ( n = 25) ( p = 0.001). ( D ) Cell clusters of 2–8 CTCs were observed in 96% of patients. ( E ) Of the recovered CTC clusters, 41% displayed a mesenchymal or epithelial-to-mesenchymal transition (EMT) phenotype (97 CTC clusters/mL) ( p = 0.007). Wilcoxon test analysis was used for comparing single vs. clusters CTCs and EpCAM+ vs. Vimentin+ CTCs. Analyses were conducted using GraphPad Prism. ( F ) Comparison of Kaplan–Meier progression-free survival (PFS) graph in patient samples ( n = 24) with a higher number of clusters than single CTCs ( n = 10) (red) and in patients who had a higher number of single CTCs ( n = 14) (black). A higher number of CTC clusters than the single CTCs was correlated with worse PFS ( p = 0.05). Log-rank (Mantel–Cox) tests were used to analyze the Kaplan–Meier PFS.

Article Snippet: The panel of antibodies (mouse anti-human CD45 (IgG2a, Biorad, Hercules, CA, USA), mouse anti-human PanCK (IgG1 Biorad, Hercules, CA, USA), anti-human EpCAM, biotinylated (goat IgG, R&D systems, Minneapolis, MN, USA), and rabbit anti-human Vimentin (Abcam, Cambridge, MA, USA) were optimized using lung cancer cell lines including H1975 and A549.

Techniques: Isolation, Immunofluorescence, Staining, Comparison