High-throughput imaging assay for evaluation of cell migration in oncology studies

Oncology Research - High-throughput imaging assay for evaluating cell migration

In studies such as metastatic tumors, inflammation, and chronic diseases, observing cell migration is an important method. The method of detecting cell migration contributes to a clearer understanding of the mechanism of the disease, and the evaluation of drugs and therapeutic effects is also extremely important. We introduce a new approach that combines the MD ImageXpress Micro high-content system with the Oris cell migration assay plate to detect cell migration.

The Oris cell migration assay plate is a consumable in the form of a 96- well plate that can be closed in the center of the plate, making it impossible to plant cells in the middle. When the silica gel block is removed, the surrounding cells migrate into the central region. At this time, fluorescent cells can be labeled with a living cell reagent such as Calcein-AM , or the cells can be labeled and labeled for analysis.

The ImageXpress Micro high-content system enables full-hole imaging, so images of the migration area in the well can be acquired at high speed and efficiency.

Introduction

The Oris cell migration plate uses a silica gel block to block the central region of each well of the 96- well plate, so that the central region cannot be planted with cells, so that a circular detection region of 2 mm in diameter is obtained at the center of the well . When the cells migrate, the surrounding cells migrate into the area. Central monitoring area. Oris cell migration plates are available in a variety of extracellular matrices, such as type I collagen and fibronectin, and are plated in 96- well plates for cell migration experiments. We used HT-1080 and MDA-MB-231 cells to study the effects of different levels of MEK inhibitor U0126 on cell migration in three Oris plates without extracellular matrix . In this experiment, we can see that the ImageXpress Micro high-content system can quickly and easily obtain images of the monitoring area in each well. The software system can automatically calculate the area occupied by the cells in the monitoring center of the hole center, so that the experimental results can be quickly obtained.

material

● Oris cell migration assay board

● HT-1080 fibrosarcoma cells

● MDA-MB-231 breast cancer cells

● HT-1080 cell culture medium: EMEM containing, antibiotics, sodium bicarbonate, sodium pyruvate, 10% FBS-temperature fire.

● PBS

● 8% glutaraldehyde

● Triton X-100

● DMSO

● MEK inhibitor U0126

● PI

● ImageXpress Micro high-content system

experimental method

1. The HT-1080, and MDA-MB-231 cells at a density of 30,000 cells / well grown to the three extracellular matrix-coated plates Oris cell migration (FIG. 1). Incubate at 37 degrees for 6 hours to allow the cells to adhere firmly.

After 2.6 hours, except control wells, silica gel Oris removable closing plate (columns 1,5,9)

3. At 37 °C, 0.1% DMSO or MEK inhibitor U0126 was added to the wells and incubated for 16 hours to allow cells to migrate freely.

4. Cells were fixed with 0.25% glutaraldehyde and disrupted with 0.1% Triton X-100 .

5. Rinse with PBS and incubate at 1 ug/mL PI for 30 minutes at room temperature . No secondary flushing is required.

6. Samples were placed in the ImageXpress Micro high-content system for cell imaging under a 2X objective and a standard Cy3 filter set. Under the 2X microscope, the ImageXpress Micro system is capable of full-hole imaging, allowing images of the cell area and detection area throughout the well to be obtained at one time.

Figure 1. Oris cell migration plates coated with three different matrices . AD was seeded with HT-1080 cells, and EH was seeded with MDA-MB-231 cells.

result

The ImageXpress Micro system captures images in the well and uses an optimized analysis system to identify fluorescent cells and identify the background in a specific area ( ROI ). By identifying the cells in the central detection zone, the number of cells migrating into the zone can be analyzed. The ImageXpress Micro system automatically captures all images in 5 minutes, and the images are automatically saved into the MDCStor database, allowing automatic analysis of all images when needed.

Figure 2. Image of the hole obtained under the 2x lens of the ImageXpress Micro system . The image on the right shows the ring detection area.

The experimental results showed that the migration of HT-1080 and MDA-MB-231 cells in the collagen-coated pores was higher than that of the uncoated or fibronectin coating. It can be seen that HT-1080 cell migration accounts for more intermediate ROI regions than MDA-MB-231 cells .

Figure 3. The results show that the two cells migrate more significantly on the collagen coated surface than the uncoated or fibronectin coated surface.

MEK inhibitor U0126 showed a dose-dependent inhibition of cells in different surface coated wells. However, inhibition in uncoated and fibronectin coated pores is more complicated.

At the same time, ImageXpress Micro can exclude cells outside the ROI in the detection area and detect only the migrating cells in the ROI during the detection process , so the detection window is large and the detection sensitivity is high.

Figure 4. Graph showing percent inhibition relative to control inhibitors. U0126 inhibits the cells in the pores of the collagen surface from being weaker than the cells in the other surface coated pores.

to sum up:

• HT-1080 and MDA-MB-231 cells showed a dose-dependent inhibition of the MEK inhibitor U0126 .

● As the results show, the ImageXpress Micro high-content system is suitable for cell migration studies. Since the cells outside the ROI region can be excluded, the detection window is large and the detection sensitivity is high.

● Oris cell migration plate is easy to use, easy to operate, and can be used for living cells or fixed cells (live cell data not shown)

DLT-Silicone

The Double-lumen Endotracheal tube (also called Double-lumen endobronchial tube or DLT) is a type of endotracheal tube which is used in tracheal intubation during thoracic surgery and other medical conditions, to achieve the selective one sided ventilation of either the right or the left lung.