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Cell Migration Experiment Service

2020-06-23
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Tumor cell migration is one of the most important features of malignant tumors. Tumor cells invade blood vessels or lymphatic vessels or body cavities from their primary site, and some cells are brought to another site or organ by blood and lymph fluid, where they multiply and grow to form The same type of tumor as the primary tumor, this process is the invasion and metastasis of the tumor.

The current methods for detecting tumor cell migration mainly include cell scratch experiments and Trans-Well cell migration experiments. Traditional cell scratch experiments require continuous observation to evaluate the effect of drugs on cell migration. Therefore, the biggest problem they face is that they cannot be guaranteed. Each observation is at the same site, and the results cannot be quantitatively analyzed, nor can the cells within the scratches migrate or proliferate. Although the Trans-Well cell migration experiment can quantitatively detect the amount of cell migration, it cannot visually observe the process of cell migration, nor can it distinguish between migrating and proliferating cells, and it is expensive, which is not suitable for large-scale screening of anti-tumor cell migration drugs .

Introduction to cell migration experiments

For cell migration and invasion experiments, the Transwell chamber was placed in a culture plate. The chamber was called the upper chamber, and the culture plate was called the lower chamber. The upper and lower culture fluids were separated by a polycarbonate membrane. The cells under study were planted in the upper chamber. The ester membrane is permeable, and the components in the lower culture medium can affect the cells in the upper chamber. Using polycarbonate membranes with different pore sizes and different treatments, co-cultivation, cell chemotaxis, cell migration, cell invasion, etc. can be performed. Various aspects of research.

transwell
transwell

The Transwell cell is placed in a culture plate. The cell is called the upper cell, and the cell is called the lower cell. The upper cell contains the upper culture fluid, and the lower cell contains the lower culture fluid. The upper and lower culture fluids are separated by a polycarbonate membrane. We planted the cells in the upper chamber. Due to the permeability of the polycarbonate membrane, the components in the lower culture medium can affect the cells in the upper chamber, so that the effects of the components in the lower culture medium on cell growth and movement can be studied. Using polycarbonate membranes with different pore sizes and different treatments, you can conduct studies in various aspects such as co-culture, cell chemotaxis, cell migration, and cell invasion.

Transwell aperture selection

※Co-cultivation experiment: When selecting the membrane pore size, the purpose of the experiment and the size of the experimental cells need to be considered. When the membrane pore size is less than 3μm, the cells will not migrate through, so if it is not involved in the study of cell movement ability, the one below 3μm should be selected , Usually 0.4μm or 3μm, for example, in the co-culture system to study the effect of cell B secretion or metabolite on cell A, you can put A in the upper chamber and B in the lower chamber.

※ Chemotaxis experiment: 5.0, 8.0, and 12.0 µm membranes are available. The cells of the upper chamber can pass through the polycarbonate membrane to enter the lower chamber. Counting the amount of cells entering the lower chamber can reflect the chemotactic ability of the components of the lower chamber to the cells of the upper chamber.

i. Cell B chemotaxis to cell A: Cell A is planted in the upper chamber, and cell B is planted in the lower chamber. The chemotactic effect of the substances secreted or metabolized by cell B on cell A can be studied.

ii. Chemotactic effect of chemokines on cells: plant cells in the upper chamber and add a certain chemokine to the lower chamber to study the chemotactic effect of the chemokine on cells.

Tumor cell migration experiment (transwell migration)

The commonly used 8.0μm (8μm pore size membrane is the most commonly used specification for invasion and transfer experiments in the literature. This time I ordered Corning’s 24-well plate transwell insert with 8μm pore size for transwell migration and transwell Invasive BD’s 8μm 24-well plate is covered with transwell chamber), 12.0μm membrane, tumor cells are grown in the upper chamber, FBS or some specific chemokines are added in the lower chamber, and the tumor cells will run to the lower chamber with high nutritional content. Counting the amount of cells entering the lower chamber can reflect the migration ability of tumor cells. FBS is the most commonly used chemokine, and this experiment is also going to choose FBS as the chemokine for migration and invasion experiments.

Tumor cell invasion experiment (transwell invasion)

Commonly used 8.0, 12.0µm membranes, the principle is similar to the tumor cell migration experiment. The upper chamber is planted with tumor cells, and the lower chamber is added with FBS or some specific chemokines. The tumor cells will run to the lower chamber with high nutritional content, but they are different from tumor cells. The difference in migration experiments is that the upper side of the polycarbonate membrane is covered with a layer of matrigel to imitate the extracellular matrix in the body. If the cells want to enter the lower chamber, they must first secrete matrix metalloproteinases (MMPs) to degrade the matrigel before they can Through polycarbonate membrane. Counting the amount of cells entering the lower chamber can reflect the invasive ability of tumor cells.

Cell migration experiment steps

1. Preparation of Transwell cell

※Corning company’s 8μm aperture glueless 24-well plate transwell insert

           i. Pre-equilibration: Add culture medium to the 24-well plate and the transwell chamber, and equilibrate in a 37°C incubator for one hour or overnight, which can enhance the adhesion of cells.

           ii. Official use: Add 0.6ml of culture medium containing 5-10% FBS to the 24-well plate, put transwell inset into the plate (using tweezers), and add 0.1ml of serum-free culture medium to the transwell insert. (Note from the operation manual: During the experiment, the height of the culture medium needs to be observed regularly, and fresh culture medium can be added when the required height is maintained)

※ BD company’s 8μm aperture 24-well plate transwell chamber covered with base glue

           i. Rehydration: take out from -20℃ and open the package at room temperature, add 0.5ml of 37℃ culture solution to the transwell chamber and 24-well plate, and then incubate in 37℃, 5% CO2 incubator 2h makes the matrigel rehydrate.

           ii. After rehydration: Carefully aspirate the culture medium, taking care not to touch the Matrigel.

2. Preparation of cell suspension

        i. Before preparing the cell suspension, the cells can be withdrawn from serum for 12-24 hours to further remove the influence of serum. But this step is not necessary.

        ii. Digest the cells, centrifuge and discard the culture solution after the digestion, wash with PBS 1-2 times, and resuspend with serum-free medium containing BSA. Adjust the cell density to 1-5×105 cells/ml. The specific cell density needs to be pre-experimented. The cell density of migration and invasion is also different (too much cell volume, too many cells passing through the membrane will be too fast. It is difficult to count if the result is counted by counting method; if it is too small, it may not be the time to detect, all the cells have passed through, so at least also to ensure that there is a certain amount of upper chamber when the sample is collected Cells are present.)

3. Inoculate cells

      i. Take the volume V1 of the cell suspension and add it to the Transwell chamber. Transwell chambers with different specifications have different requirements for the amount of cell suspension, please refer to the instruction manual. The volume added to Cornwell’s 24-well plate transwell insert is 0.1 ml, and the volume added to BD’s glued 24-well plate transwell insert is 0.5 ml.

      ii. The volume of V2 medium containing FBS is generally added to the lower chamber of the 24-well plate (FBS concentration needs to be pre-experimented). The amount of different culture plates has different requirements. For details, please refer to the instruction manual. Special attention: air bubbles are often generated between the lower culture medium and the chamber. Once the bubbles are generated, the chemotaxis of the lower culture medium will weaken or even disappear. Special care should be taken when planting the plate. Once the bubbles appear, the chamber should be lifted and removed Bubble, and then put the chamber into the culture plate.

      iii. Routine culture for 12-48h, the time point selection should consider the cell invasion ability, chemotaxis factor and cell number, etc.

      iv. Matters needing attention

       l Need to consider whether the conversion of oligo will affect the cell proliferation and apoptosis compared with NC. If the conversion of oligo can inhibit cell proliferation and promote apoptosis, then it is necessary to pay attention to the decline of MDA-231 invasion ability. Is it because the number of cells is reduced or because the ability to metastasize and invade is reduced (do you need to add a control group that does not turn?)

l It is not allowed to be too long. Similarly, it is not too short, because there will be a certain amount of MMPs stored in the cell, and the invasion ability may not change much in a short time. At the same time, the transfer from oligo to play a role, affecting the expression of MMPs, and finally released into the culture medium, it needs a process, so the effect of too short time is not obvious, but the extension of time can not affect the changes in cell proliferation.

l The shape of the cell in the cell may not be the shape of the normal adherent culture, but it is round, and it is still in the form of suspension, but it is normal to gather into clusters (other people’s experience, pay attention to observation)

l During the cultivation process, a small amount of small bubbles will gradually form under the membrane. This is a normal phenomenon and can not be treated, but if large bubbles appear, they must be removed, otherwise the experimental results will be seriously affected. It is best to take the culture plate out of the incubator 1-2 hours after seeding the cells and make sure that no large bubbles are generated.

4. Result statistics

※Remove the base layer glue and the cells that have not invaded the glue (refer to the operation manual of BD)

      i. Remove the culture medium in the transwell chamber

      ii. With a clean wet cotton swab, gently but uniformly wipe the matrix glue on the upper surface of the cell membrane and the cells that have not invaded the gel (invasion), or the cells that have not transferred across the membrane (migration).

      iii. Repeat once with the second wet cotton swab

Note: The entire process should be completed as quickly as possible to prevent the cells on the lower surface of the membrane from drying out.

※Cell fixation and staining

      i. Crystal Violet Staining Method: Fixing first and then staining. The fixing methods in different literatures are different. There are roughly the following types: 100% methanol fixation for 10min, 4% paraformaldehyde fixation for 15min or 100% ethanol fixation for 10min. After fixation, stain with 0.1% crystal violet for 30min. This method is the most commonly used method in the literature (order crystal violet reagent)

      ii. Wright’s staining method: the same is fixed first, you can use 100% methanol for 10min, and then use the Wright’s dye solution, that is, A volume is mixed with B volume 2 times the volume and stained for the corresponding time. This dyeing method is rarely used in the literature, so the dyeing time needs to be explored. According to Brother Zhao’s previous experience, it should be more than 20 minutes.

      iii. Use BD’s Diff-Quik staining kit: This method is also used in the literature. The operation steps are strictly in accordance with BD’s manual, but it is more expensive to purchase BD’s kit.

※ cell counts

     i. Use a sharp blade to separate the membrane from the chamber. Specifically, insert the blade tip into the membrane and the edge of the chamber to form a small opening, and then rotate the chamber against the direction of the blade, so that the membrane is separated. When there are a few connections, use tweezers to clamp the membrane with the smallest possible contact area, and finally separate the remaining membrane.

     ii. Prepare a slide in advance, drop a drop of oil on it, place the split membrane with the cell side up and place it on the oil with tweezers, then add a drop of oil on the membrane, and cover the coverslip, Take a picture under the microscope and count.

Note: The cell count can be taken with a microscopic photographic device to take a few representative fields of view and then counted, or it can be counted directly under the microscope, the former is better.

The above is about the content of cell migration experiment service, cell migration and invasion experiment, the content comes from the official website of Medicilon.

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