The overall goal of this procedure is to capture the formation of lc three positive Auto Phagosomes in conjunction with Microsomes and lysosomes. This is accomplished by using HEC 2 9 3 T cells that stably expressed GFPD FCP one A marker for omega zones and transecting them with Ccfp lc three one and marking the lysosomes with red lyo tracker. The second step is to prepare the incubation chamber.
Next, the incubation chamber is placed on the microscope stage and the rich medium is replaced by starvation medium to induce the autophagy response. The final step is to select the appropriate cells and set the parameters of the video microscopy. Ultimately, live cell imaging is used to show that the lc three positive phagosomes originate from the DFCP one positive omega zones, and they eventually fuse with the lysosomes.
The main advantage of this technique over existing methods like fixed cell imaging, is that we can carefully monitor the progression of events in the same cells over time. This method can help answer key questions in the PHA fail, such as the cause of events leading to the focus formation. This study utilizes a HEC 2 9 3 T cell line that stably expresses GFPD FCP one A marker for omega zones, which are precursor structures leading to autophagosome formation 48 hours prior to live cell imaging.
See a low passage number of the cells on 22 millimeter round cover slips in DMEM culture overnight at 37 degrees Celsius in 5%carbon dioxide to a co fluency of 30 to 40%on the following day. Prepared the transfection complex mix for each plate containing optimum one, reduced serum medium extreme G nine DNA, transfection reagent, and P-E-C-F-P lc three plasma DNA lc. Three is the protein of interest whose contribution to autophagosome formation will be analyzed in this experiment mixed gently by pipetting up and down, and then incubate for 15 minutes at room temperature.
Next, aspirate the medium from the plates of cells and add fresh DMEM prewarm to 37 degrees Celsius. Add the transfection complex to cells and incubate cells at 37 degrees Celsius, 5%carbon dioxide for 24 hours. 24 hours later, the cells should be 80%confluent and ready for live cell imaging if using an organelle marker.
In the experiment, remove a two milliliter aliquot of a previously prepared mito tracker or lyo tracker containing medium and warm it to 37 degrees Celsius. In this experiment, red Lyo tracker will be used to mark the lysosomes aspirate medium from transfected cells. Replace with the lyo tracker containing medium and incubate cells for 30 to 60 minutes.
To begin this procedure, clean metal and plastic O-rings with 75%ethanol and apply silicone grease on the rim of the metal O-ring. Using forceps, remove the cover slip from the plate and dry the excess medium from the bottom side of the cover slip. This is to avoid mixing too much medium with the grease, which will increase the likelihood of leakage.
Leave the cover slip to rest on the ledge of the plastic O-ring and fit the metal O-ring on top of the plastic O-ring with the cover slip sandwiched in between. In order to create a closed chamber, top up the chamber with the medium from the plate From this point on, avoid prolonged incubation of the cells in DMEA medium without a buffering agent to prevent changes in pH. Next, put the incubation chamber on the microscope stage to starve the cells and induce the autophagy response.
Aspirate the complete medium and wash twice with two milliliters of starvation medium. After the second wash, add two milliliters of fresh starvation medium and set the timer on an appropriate imaging system configured for live cell widefield epi fluorescence is required for this experiment. This system will typically comprise a research grade inverted microscope frame, an intense broad spectrum light source mirrors and filters specific for the fluorescent proteins or dyes of interest, a high quality objective lens, a sensitive C-C-D-S-C-M-O-S camera and an incubation chamber.
The most important factor is to use a system of high sensitivity so that expression levels of the fluorescent reporters can be kept to a minimum in terms of selecting appropriate cells to image large and flat cells will allow more autophagosome formation events to be captured. In addition, select cells that have already started producing a greater number of omega zones. Use a high magnification lens to prevent photobleaching, adjust the intensity of excitation light to 10 to 20%of maximum.
Set the camera to 100 to 500 millisecond exposure, two by two spinning and 100 gain. Set the image acquisition rate to one frame every 10 seconds. Start the video capture after 30 minutes or well into the autophagy response in order to capture a larger ratio of autophagosome formation events per video.
In this study, cells expressing G-F-P-D-F-C-P one and CCF PLC three were starved for 30 minutes and then imaged at rate of one frame every 10 seconds. This video shows an autophagosome formation event. The playback rate is four frames per second.
A montage of the representative autophagosome formation event captured in the video is shown in this figure signals from the green channel indicating DFCP one are pseudo colored green and signals from the blue channel indicating lc three are pseudo colored red. The formation of an omega zone becomes apparent from the second frame in the form of a small spot indicated by the arrow. The omega zone starts expanding in order to form the characteristic ring-like structure and reaches its maximum diameter after approximately six minutes.
Next, the omega zone starts collapsing and eventually disappears after approximately 10 minutes. When the formation of the lc three positive structure or autophagosome is placed in the context of the Omega zone formation, it was observed that the autophagosome appears after the omega zone and becomes clearly visible. After approximately one and a half minutes, the autophagosome starts expanding in close association with the Omega zone spot and then ring when the omega zone starts collapsing, the autophagosome buds off.
Eventually, the autophagosome stays behind apparently to fuse with the lysosomes after the omega zone disappears. In this next video, a lysosome tracker indicated in blue was added in order to capture the temporal and spatial association of the forming autophagosome. With lysosomes, the playback rate is four frames per second.
The arrow indicates first the formation of the omega zone and second, the fusion of the autophagosome with the lysosome. This figure is a montage of the events captured in the video as indicated by the arrowhead. A few minutes after the collapse of the omega zone, the nascent autophagosome starts associating with lysosomes and eventually it fuses with one lysosome fusion of the autophagosome with the lysosome, largely quenches CFPL C3 fluorescence, presumably due to the exposure of the CFP tag to the acidic environment of the lysosome.
However, the same kind of analysis can sometimes produce uninterpretable results due to a variety of reasons. In this example, the results become uninterpretable due to a drift in focus. An analysis of the images shows that the formation of an autophagosome is successfully captured by video in the beginning.
However, a drift in focus occurs after the three minute mark. The video capture continues out of focus for the next six minutes, but eventually the focus is manually corrected after the nine to 10 minutes mark. Unfortunately, this analysis makes it impossible to discriminate whether the autophagosome formation event captured when the focus is corrected is the initial event which is almost completed, or a new one that started after the drift in focus.
To prevent a drift in focus, the video should be constantly monitored and corrections made as needed, either manually or by using the autofocus feature of the microscope if available. Once mastered, this technique can be done in no more than one hour per film video if it is performed properly. After watching this video, you should have a good understanding of how to set up a microscope for live cell imaging applications, including following the formation of Autophagosome with Microsomes and lysosomes.
