high concentration matrigel  (Yeasen Biotechnology)

Journal: Cell Death & Disease

Article Title: Dopamine D 2 receptor agonists abrogate neuroendocrine tumour angiogenesis to inhibit chemotherapy-refractory small cell lung cancer progression

doi: 10.1038/s41419-025-07693-y

Figure Lengend Snippet: a , b Conditioned medium from human endothelial cells treated with a D 2 R agonist promotes apoptosis of SCLC organoids. a The SCLC PDX JHU-LX33R was suspended in Matrigel and plated at a density of 1 × 10 5 cells in 50 µL. Complete growth medium was added to the PDXs, which were grown in a humidified chamber at 37 °C supplied with 5% CO 2 . After 14 days, the Matrigel domes were dissolved by adding dispase (1 U/mL) containing cold complete medium, followed by TrypLE, then the cells were replated at a density of 10 4 cells per well of a 96-well plate in Matrigel and topped off with 100 µL conditioned medium harvested from HUVEC that had been stably transduced with plasmids encoding control LacZ-specific shRNA ( shControl CM ) or D 2 R-specific shRNA ( shD 2 R CM ) and subsequently treated with a D 2 R agonist (quinpirole). b Conditioned medium collected from shControl HUVEC treated with the D 2 R agonist quinpirole (50 µM) increased the caspase-3-mediated apoptotic response in the SCLC chemotherapy-resistant organoid model. c , d Treating human SCLC PDX organoids with D 2 R agonist quinpirole contributes to an enhanced CD8 + T cell response in the SCLC immune microenvironment. c JHU-LX33R SCLC PDX organoids were treated with D 2 R agonist quinpirole (50 µM) for 72 h, and conditioned medium from the organoids was collected and placed on human CD8 + T cells. d Human CD8 + T cells were cultured in growth medium containing 100 IU/mL IL-2 ( IL-2 : negative control) or a T cell activation cocktail of 100 IU/mL IL-2, CD3 antibody, and CD28 antibody ( Activation : activation cocktail only; Vehicle CM : activation cocktail and vehicle-treated SCLC PDX conditioned medium; Quinpirole CM : activation cocktail and 50 µM quinpirole-treated SCLC PDX conditioned medium; Quinpirole: activation cocktail and 50 µM quinpirole). The human T cells were subjected to flow cytometry using fluorophore conjugated antibodies to detect granzyme B and results have been plotted in a bar graph. Micrograph (right of the bar graph) showing the gating strategy for identification of CD8 + T cells expressing Granzyme B. Two independent biological repeats and 4 technical repeats were performed. One-way ANOVA followed by Tukey’s multiple comparisons test was performed.

Article Snippet: One million luciferase-labelled human DMS-53 SCLC cells suspended in 80 μL PBS (Corning) and high-concentration Matrigel (Corning; Cat. no.: 354248) were orthotopically injected into the left thoracic cavity of 8- to 12-week-old male and female mice anesthetized with pharmaceutical-grade ketamine (90–120 mg/kg) and xylazine (5–10 mg/kg).

Techniques: Stable Transfection, Transduction, Control, shRNA, Cell Culture, Negative Control, Activation Assay, Flow Cytometry, Expressing