Cell invasion is an important step in cancer progression and angiogenesis. In vitro invasion assays typically assess the ability of cells to break down reconstituted basement membrane, such as BD matrix, gel matrix, and migrate through cell culture inserts. The tumor invasion assay begins when cells are added to Fluor block inserts coated with matri gel.
A reconstituted basement membrane chemo attractant is added to the basal chamber and the system is incubated. Overnight invasive cells will be able to degrade the matri gel barrier and pass through the flora block membrane, a specialized membrane that protects Fluor from ambient light. The contents of the apical chamber are discarded and the multi-well insert is added to a second plate containing a Fluor.
After incubation, viable cells are stained and can be detected both visually and by a microplate reader. Hi, I'm Jeff Partridge from BD Biosciences Discovery LabWare. Today we'll show you a procedure for floor block tumor invasion assay.
We use this procedure to study the invasion of tumor cells through a basement membrane towards the chemo attractant. So let's get started. Grow the cells for the tumor invasion assay to about 80%confluence.
In this experiment, we will be using HT 10 80 and N NIH three T three cell lines. HT 10 80 cells are proven to migrate and invade the matrigel barrier in the system and hence serve as good positive controls for the assay. NIH three T three cells are not invasive, but do migrate and hence serve as good negative controls for this procedure.
To begin, the assay rehydrate the system by removing the BD bio coat tumor invasion system Package from minus 20 degrees Celsius storage and allow it to come to room temperature. Since the assay requires sterile technique, let the insert come to room temperature in the tissue culture hood. Once the packages of room temperature, open it to rehydrate the basement membrane through which your cells of interest may pass.
At 500 microliters of warm medium to the interior of the insert wells, allow the plate to rehydrate for two hours at 37 degrees Celsius, 5%carbon dioxide. There is no need to rehydrate the uncoated BD Falcon Flora Block 24 multi-well insert plate as it will be used as a cell migration control to prepare the cells for the invasion assay. Trypsin is the cell mono layers and resus suspend the cells in serum free DMEM at five times 10 to the four cells per milliliter.
If a different cell type is used, the optimal seeding density must be determined empirically. After rehydration of the coated plate, carefully remove the medium from the insert wells without disturbing the layer of MA gel on the membrane. The system is now ready to be used.
Do not let the MA gel layer dry out. To begin the cell invasion assay, add 500 microliters of each cell suspension to the apical chambers of the tumor invasion system in both the matrigel coated and uncoated configurations. Next, using the sample ports for access, add 750 microliters of chemo attractant to the basal chambers within both coated and uncoated plates.
It is extremely important not to introduce any bubbles while filling the basal chambers. As a migration control, add each cell suspension as well as the chemo attractant to the uncoated BD Falcon Flora Block 24 Multi WA insert plate. Incubate the Tumor invasion System and the uncoated Falcon Flora Block 24 Multi WA insert plate for 20 to 22 hours at 37 degrees Celsius, 5%carbon dioxide atmosphere on the following day.
When the incubation is complete, the cells are ready to be labeled with a fluorescent dye calcium am. Carefully remove the medium from the apical chambers by flicking the contents into a waste container. Then immediately transfer the insert system into a second 24 well plate containing 500 microliters per well of four micrograms per milliliter.
Calcium am in Hank's balanced salt solution. Repeat these steps with migration control. Incubate the plates for one hour at 37 degrees Celsius, 5%carbon dioxide.
Do not touch the bottom surface of the insert system. At the end of the one hour incubation with calcium am the fluorescence of the invading cells can be measured. It is of utmost importance that the insert systems are red using the correct plate map.
Proper plate orientation is with well A one at the top left corner and the BD falcon logo oriented to the right as the plate is inserted into the reader fluorescence is read at wavelengths of 494 517 nanometers on a bottom reading fluorescent plate reader. You should verify your results with an inverted fluorescence microscope to ensure the plate reader data matches the visual results. These are typical plate reader data from a coated plate and an uncoated control plate obtained from cell invasion assays of the two cell lines HT 10 80 and NIH three T three.
Data from wells with no cells represent the background that may be subtracted prior to the calculation of percent cell invasion. Calculate percent invasion use the following equation. Percent invasion equals the mean relative fluorescent units or rfu of cells that have passed through the matrigel coated membrane towards the chemo attractant.
This is divided by the mean RFU of cells that have migrated through the uncoated VD flua block membrane towards the chemo attractant. This value is then multiplied by 100. This graph shows comparison of the percent cell invasion between HT 10 80 and NIH three T three cells, and as you can see, HT 10 80 cells are invasive while NIH three T three cells are not.
When the cells are checked under an inverted fluorescent microscope, the results are consistent with those obtained from the plate reader. The invasive HT 10 80 cells invaded through the matrigel as well as migrated through the uncoated membrane. In contrast, the non-invasive NIH three T three cells did not invade through the matrigel, but migrated through the uncoated membrane.
We've just shown you how to perform a floral block tumor invasion assay. When doing this procedure, it's important to keep the plates horizontal and minimize formation of bubbles. These could affect your results.
So that's it. Thanks for watching and good luck with your experiments.
