在体外细胞迁移和侵袭实验

Cell migration is a highly dynamic process. It is important for several physiological functions such as normal embryonic development, the immune response, as well as disease processes. For example, cancer metastasis and inflammation.

The study of cell migration is important for a wide range of biomedical sciences such as cancer biology, immunology, cell biology, and developmental biology. Cell migration may be divided into four mechanically separate steps od extension, formation of new adhesions, cell body contraction, and lastly, tail detachment. Here we'll describe two simple methods to carefully study the dynamic nature of cell migration in vitro.

The first method is a cell culture of enclosure assay. This assay is commonly used and highly accessible to the labs with the most basic lab setups. The cell culture of enclosure assay is an assay where using a pipette tip and scratches generated onto a confluent monolayer of cells, the confluent cell monolayer will over time close the wound that was made previously.

The dynamics of cell migration can be studied in detail using time ops photography or using snapshot pictures. The distance may be measured and over time the velocity at which the cells moved may be calculated. The second related method, the transwell cell migration and invasion assay is also highly accessible in following the plating of cells onto a transwell membrane.

Also commonly known as a boyden chamber. Their migration abilities toward chemo attractant gradient may be measured. Both of these highly accessible experimental procedures are extremely invaluable in many areas of research.

Chapter two, cell Culture Enclosure, assay. Hello, my name is Calvin Justice. I'm a graduate student at Dr.Yang's lab from the Department of Oncology at the Brody School of Medicine at East Carolina University.

And today I'll demonstrate two procedures that measured the migration and invasion capabilities with cells in vitro. Let us now begin the cell culture enclosure. Essay The cells used in the cell culture enclosure.

Assay may be grown in tissue culture plates using conventional cell culture techniques. To begin aspirate the growth medium that is used to grow the cells wash once with PBS, then add two milliliters to 0.25%tripsin EDTA to the plate and insert it into the incubator for three minutes. Remove the plate from the incubator and add three milliliters of DMM supplemented with 10%FES.

Remove the media from the plate and put it into a 15 milliliter conal tube. Centrifuge for five minutes of 1500 RPMs. After centrifugation, re suspend the cells in two milliliters of DMM supplemented with 10%FES.

Then count the cells with a hemo cytometer. When finished counting plate a number of cells that will become confluent within 24 hours. Put the plate into the incubator for 24 hours to allow cell attachment and spreading.

Remove the plate from the incubator and look at it underneath the microscope to make sure the plate is 100%confluent. Next in the biosafety hood, using pipette tip carefully without damaging the tissue culture plate, make a wound from the top of the well to the bottom. After generating the wound with the pipette tip, aspirate the media and gently wash with two milliliters of growth medium.

In this case DMEM supplemented with 10%FES to wash away any existing debris, once again, aspirate the media. Finally, add two milliliters of growth media and if available, insert the play into a temperature and CO2 controlled chamber underneath the microscope. A time-lapse photography capability in the case.

The time-lapse capability is not available. Take initial picture of the mark location and the well and put the plate into the incubator. If time-lapse photography is available, set up the time-lapse capabilities on the microscope.

We'll set our time lapse for 16 hours. With a picture taken every five minutes after 16 hours, the plate may be removed from the chamber and the pictures that were acquired over the duration of the experiment may be saved onto a flash drive. Insert the USB into the computer and open the Image J Program.

Click file. Import an image sequence. Find the images that were taken during the experiment.

Click the first file and click open. Next sequence options will appear. Pick the first picture in your list of photos and insert the increments you'll need for the video.

Now you'll see the a VI reader options. Choose what best to choose, experiment, and click.Okay. The Image J program will now make a video, save the video, click file, save as an a VI or any other option that may suit your experiment.

The video may then be analyzed in the cell study for migration features that may be of interest. Possible features that may be studied are La Malo Podal extension, cell body contraction and tail detachment. In the absence of time loss photography, the plate that was placed into the incubator for 12 to 16 hours following the initial picture can be removed.

The area that was initially photographed should be photographed again and a scale bar must be added for further measurements. After measuring the initial width of the wound that was generated in the first photo, the width measured in pixels should be converted into micrometers using a scale bar. Next, the final pictures wound width should be measured in pixels in the same area as before, and again, it should be converted into micrometers using the scale bar.

The final step is subtracting the final width from the initial width to get the total width the wound closed within the allotted time period. The final migration distance measured should be divided by the allotted time to the velocity of enclosure and micrometers per hour. The snapshot method can be repeated in numerous wells in place of varying size for a more quantitative analysis of cell migration, chapter three, trans Well Cell Migration and Innovation Assay.

To begin, you must first grow the cells of interest in a tissue culture plate for the procedure. Next, wash once with PBS and detach the cells of interest from the tissue culture plate with the cell disassociation, buffer, or trypsin. Also, we suggest you read the literature behind how trypsin may affect the cells you are studying cause it's use may affect your results.

After detachment centrifuge of 1500 RPMs for five minutes, remove and aspirate existing media, then resuspend cells and 0.1%BSA. Open the package of the Transwell insert and place it into the well designed for its particular size. If you're performing the Transwell invasion assay first, add matrigel to the top of the insert and incubate for 30 minutes before proceeding to the next step.

Plate 100 microliter of cell solution on top of the transwell membrane. Incubate for 10 minutes to allow the cells to settle down. The number of cells plated depends on the cell type and subsequent experiments may be needed.

To determine this number, use a pipette to carefully add the desired chemo attractant under the insert to the bottom of the well. Try not to remove the insert. Incubation time is cell type dependent.

Remove the transwell insert after incubation and use a cotton tip applicator as many times as needed to gently remove the excess media and cells that have not migrated into the membrane from the top of the membrane. Be careful not to damage the membrane. Add 600 to 1000 microliters of 70%ethanol into the lower chamber of an empty.

Well place the ole insert into the well with 70%ethanol and incubate for 10 minutes to allow for cell fixation. Again, use a cotton tip applicator as many times as needed to remove the remaining ethanol in cells that have not migrated into the membrane from the top of the membrane. Add 600 to 1000 microliters up to 0.2%Crystal violet to a well and position the insert into it for cell staining.

Incubate for 10 minutes. Generally remove the excess crystal violet dye from the top of the membrane. Then very carefully to avoid washing off migrated cells, dip the membrane into the distilled water as many times as needed to remove the remaining crystal violet.

Allow the membrane to dry to view the cells using a microscope. The membrane may be carefully removed with the scalpel and placed on a glass cover slip, or you may view the cells with the inserts still in the will. Using an inverted microscope, a picture should be taken of several fields of view.

To get a good representation of the number of migrated cells, the number of cells may be counted Using Adobe Photoshop counting tool. The data that is accumulated may be inserted into a bar graph for the best representation of your results. Chapter four, representative results.

Next, we'll take you through some representative results from the cell culture of enclosure assay and the transwell cell migration assay. Two related methods to examine cell motility as described in this video, we'll begin with the cell culture of enclosure assay. Here we display snapshot pictures that were taken at 0 4 8 in 12 hours.

As you can see, after the generation of the wound pictures may be taken and over time the distance of wound closure can be measured. We display our data using a scatterplot and by using the method previously discussed, we determine the velocity of wound closure over time. Furthermore, individual cell migration may also be studied in detail by using time-lapse photography.

In this case, we zoom into individual cells during wound closure, track their migration over time and observe their movement Features such as a leading edge extension, tail retraction, and general movement may be examined in detail. You'll now go over some results of the transverse cell migration assay. Here we display two pictures that were taken of the membrane and following the transwell cell migration assay.

The first picture displays the results without chemoattractant where there was little to no cell migration through the membrane. The second picture displays the results with chemoattractant where there was increased cell migration to the membrane. To the right, we display the quantification results of our experiment using a bar graph with error bars that represent standard error.

Chapter five, conclusion the data that is accumulated following the cell cultural enclosure assay in the transverse cell migration or invasion assay reveals the migration abilities of cells in a 2D environment or a 3D environment. These results may be compared to other results to potentially reveal data that was previously unknown and it also may correlate with other biochemical tests. These two related commonly used tests are valuable and easily accessible to the most basic cell biology lab setups.

We thank you for joining us today and wish you good luck on your future scientific endeavors.