Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: A) Overview of experimental design. B) Protein stain for TP63 (green), EGFR (pink), F3 (white) and DAPI (blue) in organoids treated for 3 days of DSA followed by 7 days of DSI. Scale bar represents 50 μm. C) EGFR/F3 and control FACS plots for 1 representative biological replicate (n=3 biological replicates). D-E) Protein staining for TP63 (red, nuclear) and quantification of TP63+ cells/total cells on sorted cells spun onto a glass slide using a Cytospin. Error bars represent the mean +/− the standard error of the mean. N=3 biological replicates, shown as 3 separate colors on the plot. F) Quantification of the percentage of cells in each organoid containing positive protein staining for canonical cell type markers in unsorted organoids versus organoids that were grown from isolated EGFR+/F3+ cells. N=3 biological replicates per group and n=3 technical replicates (individual organoids) per biological replicate. A total of 198 cells were counted for the EGFR-/F3- group, 110 cells counted for EGFR+/F3- group, 38 cells counted for the EGFR-/F3+ group, and 88 cells counted for the EGFR+/F3+ group. G) Protein staining of organoids derived from EGFR/F3 sorted cells for secretory marker SCGB1A1 (pink), multiciliated markers AcTUB+ and FOXJ1+ (white), epithelial marker KRT8 (green), basal cell marker TP63 (pink), goblet cell marker MUC5AC (white) and DAPI (blue). Data shown from a single biological replicate and is representative of N=3 biological replicates. Scale bars represents 50 μm. H)GFP (green), RFP (red) and brightfield images of whole organoids 11 days after re-plating and mixing EGFR/F3 sorted cells from GFP and RFP expressing groups. N=1 biological replicate with n=6 technical replicates (wells of mixed organoids). Data shown from a single experiment and is representative of n=3 experiments. Scale bar represents 200 μm (top row) and 100 μm (bottom row).

Article Snippet: Human anti-EGFR-APC (FACS) , Milteny , 130-110-587.

Techniques: Staining, Control, Isolation, Derivative Assay, Marker, Expressing

Journal: Developmental cell

Article Title: In vitro and in vivo development of the human airway at single cell resolution

doi: 10.1016/j.devcel.2020.01.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human anti-EGFR-APC (FACS) , Milteny , 130-110-587.

Techniques: Transduction, Control, Virus, Plasmid Preparation, Recombinant, RNAscope, Multiplex Assay, In Situ Hybridization, Generated, Software

Journal: Frontiers in Oncology

Article Title: Detection of EGFR Mutations in cfDNA and CTCs, and Comparison to Tumor Tissue in Non-Small-Cell-Lung-Cancer (NSCLC) Patients

doi: 10.3389/fonc.2020.572895

Figure Lengend Snippet: Assessment of different blood collection tubes for EGFR assays on cfDNA and CTC DNA after 24 and 48 h of blood storage. (A) Overall performance of the different BCTs. LBgard is identified as the preferred BCT as opposed to EDTA, which performance significantly worsens over the storage duration. (B) Initial blood cell viability in the plasma-depleted blood sample, before VTX-1 processing. (C) Presence of numerous DAPI + debris, i.e., nucleus from dead cells, in the VTX-1 output from EDTA BCTs after 48 h. (D) cfDNA yield from the plasma workflow. (E) Cell DNA yield from the cell workflow after VTX-1 processing. (F) EGFR mutation detection for cfDNA and CTC DNA, at Day 0, 1, and 2. All pictures are original.

Article Snippet: After a centrifugation (600 g , 1 min, RT) and aspiration of the supernatant, cells were fixed with 2% PFA (Electron Microscopy Sciences #157-4) for 10 min, permeabilized with 0.2% volume/volume Triton X-100 (Research Products International Corp) and 5% Goat Serum (Invitrogen) for 7 min, blocked with 10% Goat Serum for 30 min, and immunostained. (i) For the experiments assessing the different blood collection tubes , immunostaining was performed using antibodies directed against cytokeratins (CK) (FITC, Clone CAM 5.2, BD Biosciences #347653; Clone CK3-6H5 Miltenyi Biotec #130080101), against CD45 (PE, Clone HI30, BD Pharmingen #555483) and counterstained with DAPI (Molecular Probes #D3571). (ii) For all EGFR spiking experiments and patient samples , cells were stained with anti-CK FITC (Clone CAM 5.2, BD Biosciences, #347653; Clone CK3-6H5, MACS Miltenyi, #130-080-101; Clone AE1/AE3, eBioscience, #53-9003-82), anti-Vimentin AF647 (Clone V9, Abcam, #195878), anti-N-Cadherin AF647 (Clone EPR1791-4, Abcam, #195186) and anti-CD45 PE (Clone HI30, BD Biosciences, #555483) and counterstained with DAPI.

Techniques: Clinical Proteomics, Mutagenesis