Journal: bioRxiv

Article Title: Highly conserved brain vascular receptor ALPL mediates transport of engineered viral vectors across the blood-brain barrier

doi: 10.1101/2024.03.12.584703

Figure Lengend Snippet: Retrogenix microarray screen. Expression vectors encoding >6000 human membrane proteins were arrayed on slides and overlaid with HEK293 cells for reverse transfection. AAV was added and cell-bound capsids were detected with an anti-AAV9 antibody. Bottom panel: Image from microarray screen demonstrating detection of virus spotted in gelatin (positive control) and VCAP-101 bound to cells expressing ALPL. b , Human ORFeome screen protocol. A lentiviral library containing 17,000 human ORFs was used to generate a stable HEK293T pool that was subjected to iterative rounds of transduction with VCAP-102-EGFP followed by sorting of GFP(+) cells. AAV9-mCherry was added to the third round of screening to identify EGFP(+)/mCherry(-) cells expressing a VCAP-102-specific receptor. Bottom panel: FACS gating strategy showing stepwise enrichment of permissive GFP(+) cells. c, Detection of ALPL by immunohistochemistry (IHC) in brain sections from human, African green monkey and mouse . d , Impact of ALPL depletion on VCAP-102 transduction. HeLa cells were transfected with siRNAs against ALPL and transduced with AAV9 or VCAP-102 containing a luciferase transgene. Luciferase data was normalized to VCAP-102 control siRNA (mean ± SD, n=3). e , Removal of cell surface GPI-AP proteins reduces VCAP-102 transduction. HeLa cells were treated with PI-PLC and transduced with AAV9 or VCAP-102 containing a luciferase transgene. Luciferase data (mean ± SD, n=3) is normalized to untreated controls. f , Plasma membrane localization of ALPL is necessary for VCAP-102 transduction. HEK293T cells were transfected with plasmid encoding an ALPL isoform defective for plasma membrane trafficking (ALPL-V2) and transduced with AAV9 or VCAP-102 encoding luciferase. 24h post-transduction a luciferase assay was performed. Data (mean ± SD, n=3) was normalized to vector control.

Article Snippet: The Retrogenix cell microarray was performed by Charles River Laboratories. cDNA expression vectors encoding over 6000 human membrane proteins, secreted or cell surface tethered-secreted proteins were arrayed across a series of slides in duplicate and overlaid with HEK293 as previously described .

Techniques: Microarray, Expressing, Membrane, Transfection, Virus, Positive Control, Transduction, Immunohistochemistry, Luciferase, Plasmid Preparation

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A) Human leukocytes/carcinoma cells are dispersed on a cell microarray chip, followed by 15 minutes' standing to allow the cells to settle down into the microchambers, and are then stained with fluorescence-labeled antibodies against carcinoma cells. (B) Fluorescence-positive CTCs are detected by using a microarray scanner with a confocal fluorescence laser. (C) The target CTCs are analyzed quantitatively at the single-cell level.

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Microarray, Staining, Fluorescence, Labeling

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A) Photo of an actual cell microarray chip. (B, C) SEM images of a cell microarray chip. The cell microarray chip comprises 20,944 microchambers in a plastic slide on a glass slide. The cell microarray chip has 112 (14×8) clusters, each having 187 microchambers. (D) Each microchamber is 105 µm in upper diameter, 50 µm in depth, and has the shape of a frustum with a 68-µm diameter flat bottom for the accommodation of leukocytes as a monolayer.

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Microarray

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A, B) Photographic light microscopic images of T lymphoblastoid leukemia cells incubated on a cell microarray chip before (A) and after (B) washing of the chip surface. (C–F) Photos of microchamber appearance after washing when T lymphoblastoid leukemia suspensions of 2.5×10 6 (C), 5.0×10 6 (D), 7.5×10 6 (E) or 1.0×10 7 (F) cells/ml were applied to the microarray chip. Concentrations of 7.5×10 6 cells/ml of T lymphoblastoid leukemia and above afforded tight confinement and formation of a monolayer in the microchambers. (Bar: 20 µm).

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Incubation, Microarray

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A, B) Photographic light microscopic images of carcinoma cells on a cell microarray chip before (A) and after (B) washing of the chip surface. (C) Carcinoma cells showed tight confinement and had formed a monolayer in the microchamber when a concentration of 7.5×10 6 cells/ml was used. (Bar: 20 µm).

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Microarray, Concentration Assay

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A, B) Photographic light microscopic images of leukocytes on a cell microarray chip before (A) and after (B) washing of the chip surface. (C) The leukocytes showed tight confinement and had formed a monolayer in the microchamber. (Bar: 20 µm).

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Microarray

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A–I) Scanned images of leukocytes/carcinoma cells on a cell microarray chip obtained with the microarray scanner. (A) Negative control (no carcinoma cells). (B, D, G) Carcinoma cells (0.01, 0.001, and 0.0001%) were scanned in 3, 9, and 64 clusters, respectively, on the cell microarray chip. (C, E, F, H, I) Magnified views of the boxed regions. Color scale represents the intensity of fluorescent emission.

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Microarray, Negative Control

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A) Scanned images of cells on a cell microarray chip, obtained with the microarray scanner. The cells were immunostained with PE-labeled anti-cytokeratin monoclonal antibody. (B) Magnified view of the boxed region. Color scale represents the intensity of fluorescent emission.

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Microarray, Labeling

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: Analysis of spiked carcinoma cells in human whole blood on a cell microarray chip.

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Microarray

Journal: PLoS ONE

Article Title: Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

doi: 10.1371/journal.pone.0032370

Figure Lengend Snippet: (A–L) Cultured T lymphoblastoid leukemia (A–F) and leukocytes isolated from whole blood (F–L) were stained with PE-labeled anti-cytokeratin monoclonal antibody (A, G) and APC-labeled anti-EpCAM monoclonal antibody (C, I). (E, K) Merged images identify doubly-positive carcinoma cells in each panel. Magnified views of the boxed regions (B, D, F, H, J, L). Scatter-plot analysis of 3 cluster areas representing 561 microchambers in a cell microarray chip .

Article Snippet: As shown in , the cell microarray chip comprised 20,944 microchambers (105-μm upper diameter, 68-μm lower diameter, 50-μm depth, and spacing as indicated) and was made from polystyrene by the Lithographic Galvanoformung Abformung process by Starlight Co. Ltd. (Osaka, Japan) .

Techniques: Cell Culture, Isolation, Staining, Labeling, Microarray