The overall goal of this Live-cell imaging technique is to follow cellular processes like the uptake and distribution of Chemotherapeutic Agents without the need for fixation. These methods can help answer key questions in the cancer field such as:how Chemotherapeutic Agents either in free form or encapsulated in nanoparticles are taken up by tumor cells. The main advantage of this technique is that we are imaging living cells, thereby preventing fixation artifacts and allowing real-time dynamic evaluation.
We first had the idea for this method when we observed that nanoparticals broke down during fixation which led to misinterpretation of the drug delivery. To assemble the imaging chamber, place a 25 millimeter cover glass at the bottom part of the chamber and screw the top part into the bottom part. Do not tighten too hard, as the glass can break.
Autoclave the entire chamber. To plate the cells, remove the imaging chamber from the sterilization bag in a Laminar Flow Cabinet. Carefully tighten the chamber and place it in a Petri Dish.
Coat the glass of the imaging chamber with one-milliliter of 0.1 percent sterile gelatin for 20 minutes at 37 degrees Celsius and five percent Carbon Dioxide. After removing the medium from the cell culture flasks, wash the cells once with 1x PBS and detach the cells using 0.25 percent Trypsin. Inactivate the Trypsin by adding Cell Culture medium.
Then, collect the cells and spin them down at 1, 100 Gs for five minutes. Re-suspend the pellet in five-milliliters of Cell Culture medium. After counting the cells as described in the text protocol, aspirate the gelatin and plate the cells in a dilution that allows for a maximum of 70 percent coverage within 24 hours.
Allow the cells to adhere at five percent Carbon Dioxide and 37 degrees Celsius for 24 hours. Perform an evaluation of the cells under a microscope for confluency, cellular morphology, and contamination. Then, switch on the Confocal Microscope, fluorescent light, and computer.
Connect the lens heater to the objective and set it to 35 degrees. Prepare the incubation unit as shown in the text protocol. Place the incubation unit on the microscope stage and connect the in and outflow Carbon Dioxide tube.
Open the main Carbon Dioxide valve and switch on the Carbon Dioxide controller. Check that the controller is set to five percent Carbon Dioxide. Proper cell culturing conditions like temperature, CO2, pH, humidity, and sterility are crucial for long term cell imaging.
So for each incubator, that need to be properly tested. Set the stage temperature to 37 degrees Celsius. Allow the incubation unit to reach the selected temperature and Carbon Dioxide percentage.
Take the imaging chamber from the main Carbon Dioxide incubator and transport the chamber in a Petri Dish to the microscopy room. Place the imaging chamber in a custom-made 35 millimeter diameter stage holder and close the chamber with a custom-made sealing lid. Open the incubation unit, and working as fast as possible to prevent the cooling down of the cells;replace the red patch with the imaging chamber and then close the unit.
Make sure that no cables are stuck between the stage and microscope. Leave the unit to acclimatize for 30 minutes. Next, open the software and switch on the laser.
Make a new database by clicking New"under the File tab. Name and save the database. Set the configuration by clicking Config"under the Acquire tab.
Select Channel Mode"and Single Track"to evaluate a single fluorophore. Select Multi Track"for several fluorophores. Then, choose the appropriate band-past filters, matching the fluorophore.
Select the bright field channel in one of the tracks. Scanning is compromised between the fluorescent signal of the compounds and the quality of the image. Overexposure, photobleaching, signal to noise ratio, the quality of the cells, the added compounds are all factors that can influence the results and need to be properly tested.
Set the scan control by clicking Scan"under the Acquire tab. Then click Mode"to set the frame size, the scan speed, the scan direction, and the scan average. Click Channels"to set the Pinhole, the Gain and Offset, and the laser power.
Save these settings by clicking Config"in the Configuration tab and save them in the Configuration database. Set the time-lapse clicking Muti TimeX"in the Macro tab. Click Single Location.
Apply the saved configuration by clicking Single Track"and scroll to the required configuration in the Configuration database. Set the time interval and the number of scans. Load the configuration by clicking Load Conf.
Name the time-lapse in the Base File Name and select the database by clicking Image Data Base"to save the time-lapse. Set a temporary folder by clicking Temporary Image Folder"Next, open the incubating unit, lift the imaging ring, add a drop of Immersion Oil to the objective, and close the unit as fast as possible. Focus and select a position in the imaging chamber.
In the Laminar Flow Cabinet, dilute free drug or nanoparticles at a concentration of five micrograms per milliliter drug or 0.05 micromoles of lipids in Cell Culture medium. Filter through a 0.22 micron Syringe Filter if the drug or nanoparticles are not sterile. Open the incubating chamber and lift the ceiling lid.
Then, remove the medium from the cells, add one-milliliter of diluted drug or nanoparticles, and close the unit as fast as possible. Focus on the cells and make a fast scan by clicking Single"in the Scan Control tab. When the bright field image shows focused cells, start the time-lapse by clicking Start Time"in the Multi TimeX Macro.
The number of repetitions and remaining time is shown in the Macro and in the images. Check regularly to see if the cells are still in focus, or use the Autofocus. The program automatically saves the time-lapse in the database.
Do not close the database while the program is running. This is a three hour time-lapse of cells exposed to free Doxorubicin. Observe the uptake and immediate translocation of red fluorescent Doxorubicin to the nucleus.
Here, cells are incubated for three hours with encapsulated Doxorubicin. The fluorescent signal is much lower and is located in the cytoplasm. The signal is hardly visible in the nucleus.
Transporting these time lapse movies to Image J, a region of interest can be drawn around the nucleus and the pixel density can be measured. The graph presented here shows the difference in nuclear uptake over time in cells incubated with either free or encapsulated Doxorubicin. Shown here, are cells incubated with encapsulated Doxorubicin, shown in red.
The purple signal represents the carrier and Lysosomes are stained with a green Live Cell marker. Comparing both cell types shows a difference in Lysosomal distribution. Lysosomes in B16BL6 cells are distributed throughout the cytoplasm.
Whereas, Lysosomes in BLM cells are located close to the nucleus. However, both cell lines show a high colocalization of red and purple signal in Lysosomes indicating that Doxorubicin and its carrier are trapped in these organelles. After watching this video, you should have a good understanding of how to image the uptake of Chemotherapeutic Agents and other fluorescent markers in Living Cells for several hours or even days.
