This demonstration uses two and 3D live cell imaging to understand the spatial temporal relationships of proteins involved in the DNA damage response. First, transfect or transduce your cell line of choice with expression constructs containing appropriate fluorescent markers. Here M Cherry and GFP next acquire a series of control videos showing the fluorescently labeled cells under normal conditions.
Then apply an appropriate treatment and capture data of the treatment effects on the fluorescent cells process and analyze the video data for spatial temporal relationships of proteins. Ultimately, live cell fluorescence microscopy can detail cellular dynamics like the formation of 53 BP one foci in response to DNA damaging agents or 53 BP one's behavior during mitosis. We first became interested in two and 3D lifestyle imaging when we wanted to study how varying treatments affected mitosis in certain cell lines.
This experimental approach allows us to study the DNA damage response and the maintenance of genomic stability more easily than traditional methods. Additionally, you can use this method to study how certain proteins interact in various signaling pathways and various cell lines. The main advantage of lifestyle imaging over traditional techniques like cyto chemistry is that it allows the study of the DNA damage response proteins in real time.
Optimizing the right recording conditions can be challenging. This demonstration can be used to see the many small steps needed to ensure success. Remember that persistence pays off when it comes to minimizing photo bleaching, finding the right recording intervals and the right recording length of time, et cetera.
Transfect or transduce the appropriate cell lines with the plasmids for mCherry and GFP fusion protein expression. Maintain the cultures under drug selection and periodically check for expression of the fluorescent proteins the day before image acquisition. Harvest the cells using trypsin.
Then seed low density cultures onto 3.5 centimeter fluoro dish glass bottom plates. This protocol was developed using the Zeiss cell observer SD spinning disc confocal microscope, equipped with an axio observer Z one stand First, verify that the CO2 gas is running to the CO2 module of the incubation system. If the microscope is supported by an anti vibration Airtable, then turn on the air supply for the Airtable.
Now switch on the power for the microscope stand spinning disc unit cameras, incubation modules, HXP illuminator, motorized stage, argonne laser, and computer. Turn on the switches for the laser lines to be used. Start up the Axio vision software.
The Avision software may be customized with windows and pull down menus that are specific to the microscope and components that it controls. As such, each user interface has a potentially unique look Approximately one hour prior to imaging. Locate the controls for the incubator and turn on the heating for the upper chamber and the stage plate.
Set the temperature to 37 degrees Celsius. Turn on the CO2 control and set the level at 5%Select the objective lens for imaging In this study. The objective lens requires use of an immersion medium that has a refractive index similar to water.
If you need to image multiple positions over long periods of time, be sure to apply enough immersion medium so that the scope does not dry out. Place the culture dish on the stage and bring the objective lens up into contact with the bottom, using either the microscope controls or the software. Direct the emitted light to the eye pieces and select the appropriate wide field filter set for the fluorescent signal of interest.
Now view through the ocular lenses. Focus the image and locate a suitable field of cells using either the software or the microscope controls. Direct the emitted light away from the eye pieces to the port with the confocal spinning disc unit in the software.
Turn on the appropriate laser for this study. For each channel, adjust the intensity of the laser by adjusting the kuo optic tunable filter control to an appropriate level. Next, select the appropriate dichroic mirror and emission filters.
Open the shutter to the spinning disc unit. Now select the live window to display the current field of view. Now open the camera control window.
Select the camera to be used and set exposure time to approximately 100 milliseconds. Adjust the percent and EM gain as necessary. Also, open the control for the confocal spinning disc unit and adjust the spinning disc speed by entering the camera exposure time that was set to capture a suitable image.
Click set to lock in the change. Now open the multi-dimensional acquisition window. Select the channel tab and load or select appropriate channels defined for the Flora Fours.
To ensure image registration, use a common dichroic mirror for both channels. Set the software to auto focus. Proceed to the MDA window and click on the ZS stack tab.
Select ZS stack at current focus position. Set the range for a 10 micron Zack and choose optimal for the number of steps to ensure nyquist sampling through Z.Now click start and analyze the resulting Z stack image. Select the time tab.
Set the interval between imaging time points and the overall duration of the session. For multi-point imaging of several cells in the dish, open the MDA window. Select the position tab and verify that the apply setting before or after time point per position is checked.
Now select mark. Find using the live view, move the dish around and select appropriate fields of view. Click start in the multidimensional acquisition menu to begin the experiment and record a control video.
Proceed to add the appropriate treatment and record the experimental video at determined time intervals for a defined period of time. For this cell line and for monitoring the entire process of mitosis, we used an interval of seven and a half minutes and it for four to five hours. You will have to determine the particular settings for your cell line and for what you want to study.
Open the velocity software, create and name a new library and import the experimental video files for viewing the files. Try using the extended focus setting, adjust the videos as necessary. Velocity is equipped with a range of tools to help improve the quality of your acquired images.
If the settings on the microscope are appropriate, it will save a lot of time later on In editing. Often it is helpful to devolve your images and adjust the brightness and contrast your specific needs will vary based on your experiment To view the cells in 3D. Switch to the 3D opacity setting.
This permits rotation of the 3D rendered cells in space, thus providing multiple perspectives of structures of interest within the cells.Movies. Still images can be exported into a variety of file types based on user preference Compared to the controls. Fibroblast cells exposed to CPT formed foci within five to 10 minutes and maintained these foci throughout the duration of recording.
Interestingly, in human embryonic kidney cells, 53 BP one dissociates from chromatin at the onset of mitosis, forming a thin haze around the condensing chromosomes. As teleph occurs and mitosis comes to an end 53 BP one, once again aggregates into distinct foci. We hope that this video will enable you to study the spatiotemporal relationships of proteins involved in the DNA damage response as well as other pathways.
For example, in the field of chromatin dynamics, this technique was used to study how 53 BP one binding affects the movement of telomeric ends of DNA. After the creation of genetically modified cells, two and 3D live cell imaging can be performed in approximately four to eight hours. Ensure that the microscope settings are adjusted appropriately to have optimal imaging.
