Journal: ACS Pharmacology & Translational Science

Article Title: Selective Elimination of Osteosarcoma Cell Lines with Short Telomeres by Ataxia Telangiectasia and Rad3-Related Inhibitors

doi: 10.1021/acsptsci.0c00125

Figure Lengend Snippet: Selective death of osteosarcoma with short telomeres following treatment with ATR inhibitor VE-822. Short telomere cell lines, OHSN, HOS-MNNG, and HAL, and long telomere cell lines, MG-63 and MHM, treated with VE-822 were assessed using IncuCyte live cell analysis. Medium was supplemented with the live-cell-impermeable dye SYTOX to quantify cell death. (a) Representative images of OHSN and MG-63 treated with vehicle (DMSO) or 1 μM VE-822 at the indicated time. A 15 μm scale bar is shown. Phase contrast images are overlaid with SYTOX death dye signal (red) marking dead cells. (b–f) Representative graphs depicting net death over time, reflected by SYTOX death dye incorporation normalized to cell density for OHSN (b), HOS-MNNG (c), HAL (d), MG-63 (e), and MHM (f). Data representative of two or more experiments.

Article Snippet: Time-lapse observations were carried out using an IncuCyte ZOOM live-cell analysis system (Essen Bioscience), as described previously.

Techniques:

Journal: STAR Protocols

Article Title: 3D in vitro culture system to study collective migration in mammary organoid epithelium

doi: 10.1016/j.xpro.2021.100778

Figure Lengend Snippet: Prepare FGF10 soaked bead and needles for migration assay (A–A'') Heparin beads of different brands. (A) from sigma company, Cat#H5263, (A′) from Abcam company, Cat#ab193268, and (A'') from ADAR Biotech company, Cat#6024. (B) A stereo microscope used to pick up suitable beads, organoids and to set up the migration assay. (C) The zoom knob was set at 3 for all steps. (D and D′) Picture of Heparin bead 100 μm (D) and 150 μm (D′). (E) A 100 μm and a 150 μm beads were sealed in PBS in a 35 mm dish. These two beads will serve as a reference for picking beads and setting up steps. (F) Picked beads in a 0.2 mL tube and suspended in 20 μL of FGF10 solution. When upside down, the solution should not move along the tube wall, yet the bead will move freely in the solution. (G) 0.2 mL tube containing the picked beads was secured in a capless 1.5 mL tube with tape. (H) the 1.5 mL tube was rotated on a mixer. (I) Steps of preparing needles for migration assay.

Article Snippet: For time-lapse imaging, DIC microscopy can be performed on a Zeiss Cell Observer SD spinning disk confocal microscope using an EC Plan-Neofluar 10×/0.3 Ph1 M27 objective lens (Item no.: 420341-9911-000), an Analy DIC Transmission light reflector, an sCMOS camera at 37°C with 5% CO 2 .

Techniques: Migration, Microscopy

Journal: STAR Protocols

Article Title: 3D in vitro culture system to study collective migration in mammary organoid epithelium

doi: 10.1016/j.xpro.2021.100778

Figure Lengend Snippet: Isolation of mammary organoids (A and A ′) Collected mammary tissue was placed in the lid of a Petri dish (A). The MG tissue was minced (A ′) with double surgical blades about 300 times. (B and C) (B) Transfer mammary gland to collagenase solution, (C) incubated at 37°C for 22 min with rotation, thereby breaking down the fat pad into relatively dispersed pieces. (C′) After digestion, DNase was added, and the suspension was pipette up and down ten times with a 10 mL serological pipette. (C'') Centrifugation separates the suspension into three layers, the top layer is an opaque fatty layer, the middle is a transparent aqueous phase, and the bottom precipitation is the organoid pellet. (D) The organoid pellet was resuspended in 4 mL DMEM/F12 containing DNase and transferred to new tubes. (D′) DIC images of the resuspended pellet with many single cells. (E) Pellet of grayish-white organoids after four differential centrifugations. (E′) DIC images of the resuspended pellet. Single cells were removed. (F) DIC image of picked organoids. The proper size is about 50 μm diameter × 100–200 μm length. (G and G′) Pictures of organoid epithelium under different focus levels. It is hard to distinguish endothelium from organoid epithelium under the stereo microscope.

Article Snippet: For time-lapse imaging, DIC microscopy can be performed on a Zeiss Cell Observer SD spinning disk confocal microscope using an EC Plan-Neofluar 10×/0.3 Ph1 M27 objective lens (Item no.: 420341-9911-000), an Analy DIC Transmission light reflector, an sCMOS camera at 37°C with 5% CO 2 .

Techniques: Isolation, Incubation, Suspension, Transferring, Centrifugation, Microscopy

Journal: STAR Protocols

Article Title: 3D in vitro culture system to study collective migration in mammary organoid epithelium

doi: 10.1016/j.xpro.2021.100778

Figure Lengend Snippet: Prepare the equipment and materials for the experiment (A) Full view of the workbench. Materials that necessary for the migration experiment are on the table. The table is set in a sterilized room. (B) Experimental materials on ice. Coated heparin beads and fresh organoids are stored in a 35 mm dish. PBS, BSA, Matrigel, and needles are store in tubes. (C–D''') Setting up of the stereo microscope during the operation of the migration experiment. (C) Align the center of the working well with one cold block hole and the stereo microscope views. (D) A blue rack was used for picking up the beads and organoids. Use the top light on the stereo microscope. (D′–D''') The black disk was removed when arranging the beads and organoids in a 24-well plate. Use the cold light source. (E and E′) Water condensation on the cold block surface (E), wipe the water off the surface (E′).

Article Snippet: For time-lapse imaging, DIC microscopy can be performed on a Zeiss Cell Observer SD spinning disk confocal microscope using an EC Plan-Neofluar 10×/0.3 Ph1 M27 objective lens (Item no.: 420341-9911-000), an Analy DIC Transmission light reflector, an sCMOS camera at 37°C with 5% CO 2 .

Techniques: Migration, Microscopy, Blocking Assay

Journal: STAR Protocols

Article Title: 3D in vitro culture system to study collective migration in mammary organoid epithelium

doi: 10.1016/j.xpro.2021.100778

Figure Lengend Snippet: Schematic diagram of the migration experiment steps (A) Organoids of appropriate size were selected under a stereo microscope and placed in a 35 mm dish for later use. (B) Side view of the process of laying Matrigel on a 24-well plate. First, place the plate on the cold block for 1 min, draw a 1 cm circle on the bottom using a 10 μL tip containing 7.5 μL Matrigel, fill the circle, then add an extra 7.5 μL Matrigel. (B ′ and B'') A top view diagram of the above process. Once Matrigel is laid, the plate is heated on the metal block at 37°C for 2 min and then cooled down on the cold block for 2 min. All subsequent operations are performed on the cold block. (C) Add 20 μL top Matrigel to the solidified coating gel. (D) Aspirate five organoids at a time with a P2 pipette setting at 1.2 μL and add them to the right side of the top Matrigel. In the same way, aspirate five beads and add them to the left side of the top Matrigel. Under the dissection microscope, line up the beads with the fine needle, >300 μm apart between beads. Then, the organoid was juxtaposed to the right side of the bead in a one-to-one manner, 50 μm–100 μm apart between the bead and organoid. (E) After completion, the overall view of the position and proportion. (F) The plate was heated on the metal block at 37°C for 8 min to solidify the Matrigel, and then 1 mL basal medium was added. (G) For time-course images or IF staining, the sample can be cultured in an incubator at 37°C. The sample can also be maintained in time-lapse microscopy. (H) Flip the image horizontally when present the data.

Article Snippet: For time-lapse imaging, DIC microscopy can be performed on a Zeiss Cell Observer SD spinning disk confocal microscope using an EC Plan-Neofluar 10×/0.3 Ph1 M27 objective lens (Item no.: 420341-9911-000), an Analy DIC Transmission light reflector, an sCMOS camera at 37°C with 5% CO 2 .

Techniques: Migration, Microscopy, Blocking Assay, Transferring, Dissection, Staining, Cell Culture, Time-lapse Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Adropin Contributes to Anti-Atherosclerosis by Suppressing Monocyte-Endothelial Cell Adhesion and Smooth Muscle Cell Proliferation

doi: 10.3390/ijms19051293

Figure Lengend Snippet: Effects of adropin on migration, proliferation, apoptosis, and ECM expression in HASMCs. ( A ) Migration was determined in 10 HASMCs per plate using a BIOREVO BZ-9000 microscope in serum-free SmGM-2 with or without AngII (500 nmol/L) and adropin (0, 100, 1000 ng/mL). Four independent experimental replicates were performed. * p < 0.0001 vs. 0 ng/mL of adropin. † p < 0.0005, ‡ p < 0.0001 vs. AngII. ( B ) The proliferation was determined by WST-8 assay following a 48-h incubation of HASMCs in 5% FBS-SmGM-2 with the indicated concentrations of adropin ( n = 5). § p < 0.05 vs. 0 ng/mL of adropin. ( C ) HASMCs were stained as apoptotic cells (green) using the TUNEL method after a 48-h incubation in 5% FBS-SmGM-2 with the indicated concentrations of adropin. Nuclei were co-stained with 6-diamidino-2-phenylindole (blue). The graph indicates the percentage of apoptotic cells ( n = 3). Scale bar = 100 μm. ( D ) HASMCs were incubated for 24 h in serum-free SmGM-2 with the indicated concentrations of adropin, and then harvested for immunoblot of collagen 1, collagen 3, fibronectin, elastin, MMP2, MMP9, and α-tubulin. Representative data showing protein expression (upper panels) with densitometry following normalization relative to α-tubulin (lower panels, n = 5–6). § p < 0.05 vs. 0 ng/mL of adropin.

Article Snippet: The migration distance was measured in 10 cells randomly chosen in each well using a time-lapse cell culture observation system (BIOREVO BZ-9000 microscope; Keyence, Osaka, Japan) [ , , , , , , , , ].

Techniques: Migration, Expressing, Microscopy, Incubation, Staining, TUNEL Assay, Western Blot