The overall goal of this demonstration is to compare three different cell proliferation assays and present the advantages and disadvantages of each method. This method can help answer key questions in the cancer research field, such as the use of the most appropriate cell proliferation method. Preparation of this method is critical, as the cell imager steps are difficult to learn.
They require you to focus on the cells before applying the appropriate cell counting mask. To begin this procedure, grow two MCF-7 cell lines for three days to 75 to 80%confluence in the T75 square centimeter tissue culture flasks using phenol red-free DMEM. After three days, remove the cells from the flasks by pouring off the medium into a waste container.
Then, immediately wash the cell layer with two milliliters of pre-warmed two times trypsin. Aspirate the trypsin, and add another two milliliters of pre-warmed trypsin to the cell layer before placing it in an incubator. Once the cells have detached, wash them with 10 milliliters of warmed fresh supplemented DMEM.
Then, transfer the cell suspension to a sterile 15 milliliter tube and centrifuge it for five minutes at room temperature. After five minutes, carefully aspirate and dispose of the supernatant. Afterward, re-suspend the cell pellet in five milliliters of fresh supplemented DMEM.
To perform a cell count, dilute 100 microliters of the cell suspension in 900 microliters of one times DPBS. Then, place a new 60-micrometer sensor onto the automated cell counter. Hold down the plunger and submerge the sensor in the diluted cell suspension.
Slowly release the plunger on the cell counter. Then, remove the sensor from the cell counter when completed. Seed the cells in a final volume of 100 microliters in a 96-well plate at about 20%confluency to allow room for cell growth and measurement of proliferation over three to five days.
To determine the cell count using a hemocytometer, pre-warm both the medium and trypsin to 37 degrees Celsius in a cell culture incubator, oven, or water bath. Next, aspirate the medium from the cells into a waste container. Then, wash them once with 30 microliters of two times trypsin and aspirate the medium into the waste container again.
Next, wash the cells again with 30 microliters of two times trypsin, and incubate them for five minutes at 37 degrees Celsius. Gently tap the plate edge to dislodge the cells. Subsequently, add 50 microliters of supplemented DMEM and mix the cells by pipetting until a single cell suspension is formed.
Place the glass cover slip over the counting chambers and affix it until Newton's refraction rings are seen between the cover slip edges and the helocytometer. Then, gently pipette 20 microliters of the cell supension under the cover slip and allow the counting chamber to be filled by capillary motion. Subsequently, visualize the counting chambers in the grid layout under 10x magnification.
In this procedure, thaw the luminescence reagent in a 22 degrees Celsius water bath for 30 minutes. Gently invert the bottle to obtain a homogeneous mix. Then, equilibrate one plate of seeded cells at room temperature for 30 minutes.
Afterward, add 100 microliters of luminescence reagent to each well. Mix the contents on an orbital shaker for two minutes. Then, allow the plate to incubate for 10 minutes at room temperature.
To set up a luminescence experiment using the multi-mode plate reader software, turn on the multi-mode plate reader and open the software. In the Task Manager, select Experiments and Create New. Then, select File from the side toolbar, and under the Protocol tab, select Procedure.
Next, select Greiner 96 flat bottom as the plate type, and check the Use Lid box. Select the Read action under the Read method box. Then, select Luminescence as the detection method and click OK.In the Read Step box, select the wells to be scanned under the Full Plate tab, and select wells to act as blank wells.
Set the Filter Set to Empty Filter. Set the Gain to 135, with an Integration Time of 5 seconds per well and a Read Height of 6.5 millimeters. Click OK to save the settings for the luminescence read.
To perform a luminescence read, eject the plate holder by selecting the Instrument Control tab and click Plate Out. Place the 96-well plate onto the plate holder, ensuring the lid is on. Close the plate holder by clicking on Plate In under the Instrument Control tab.
Then, click Read Now in the toolbar to perform a luminescent read. After that, export the RLU measurements for each well to a spreadsheet for further analysis. To set up a cell imaging experiment, turn on the multi-mode cell imager and open the software.
In the Task Manager, select the Experiments tab and Create New. On the File toolbar, click on the Protocol tab and then the Procedure tab. Select the Set Temperature action.
Set the Incubator to On, and set the Temperature to 37 degrees Celsius. Check Preheat, then click OK to save the settings. Next, select the Read action.
Click on Image as the detection method. Then, select the Endpoint/Kinetic read type and the Filters optics type, and click OK.After that, click the Full Plate tab, and select the wells on the plate to be imaged. Click OK to save the settings.
Now, select the 2.5 times objective from the drop-down Objectives option. Under the Channels tab, select GFP 469.525 and Bright Field. Check Auto Exposure for both channels, and select the auto exposure well.
To determine auto focus settings, click Options. For the auto focus options, select the method Scan and then auto focus. Click OK to save the settings.
After that, set the horizontal and vertical offset from the center of the well to zero. Select to scan multiple images per well in a three by two montage. Then, click OK to save the procedure settings.
To read the experiment on the selected plate, click on the Instrument Control tab and select the Plate Out function. Place a 96-well plate on the plate holder, ensuring the lid is on. Under the Instrument Control tab, select the Plate In function.
Next, select Read Now from the plate tab, and repeat the read every 24 hours, up to 96 hours post-seeding. To analyze an image, click on the Data tab on the imaged plate and select Picture GFP 469, 525 plus Bright Field. Then, double-click on the imaged well.
Now, click on a loaded image. Select Analyze, and then select the single image of the montage to be analyzed. Next, click OK.Afterward, check the GFP channel only, and set the parameters as outlined in Table 3.
After that, click Start to the apply the parameters to the imaged cells. Observe the cell counting mask placed over the imaged cells, which is used to determine the cell count per image. Click Apply Changes, and maintain these settings for each plate imaged throughout the experiment.
In this figure, a linear regression analysis was performed to compare the correlative relationships between the different methods examined for measuring the cell proliferation in MCF-7-LeGO cells and MCF-7-delta 40p53 cells. A Pearson's correlation coefficient was calculated, and the significance was determined between the different methods measuring cell proliferation. The strongest correlation was observed between the comparison of the luminescence-based assay and the cell imager.
Shown here are the representative images of GFP positive MCF-7-LeGO and MCF-7-delta 40p53 breast cancer cells captured using the cell imager every 24 hours until the cells reach close to 100%confluence. This method provides useful cellular information, including being able to visually monitor cell growth across multiple days, and compare cell size and cell morphology between different cell lines. A summary of the advantages and disadvantages of each method tested is shown here, which demonstrates the versatility of each of the methods and will assist readers in choosing the most appropriate method.
After watching this video, you should have a good understanding of three different cell proliferation assays, and should be able to determine which one best suits your own experimental design.
