Live cell imaging is particularly useful when studying the dynamics of organal trafficking. This video will highlight a procedure used to image dense core les in cultured hippocampal neurons. Transfection complexes are first obtained by mixing D-N-A-M-E-M medium and the lipectomy transfection reagent.
Since hippocampal neurons are cultured on inverted cover slips facing a glial monolayer, these cover slips must be turned over in order to expose them to the transfection reagent. After allowing ample time for expression of GFP tag dense core viscal proteins, an imaging chamber is constructed. An imaging of dense core ICALs in cultured neurons can be performed using wide field fluorescence microscopy.
This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes. Hi, I am Michael Silverman and welcome to my laboratory for cellular neuroscience at Simon Fraser University. We're interested in how organelles move in nerve cells.
To approach this question, we label membrane don organelles, specifically dense core vesicles in cultured hippocampal neurons. And to show you how we do this, I'm going to turn it over to David. Hi, I am David from the Silverman lab.
Today I'll show you a procedure for live cell imaging of dense core vesicles and cultured neurons using widefield western microscopy. Let's get started. For most experiments, we use dishes of rat hippocampal neurons that have been cultured at a cell density of 250, 000 cells per six centimeter dish for each transfection labeled two 1.5 milliliter tubes, one for the plasmid, DNA and the other for the transfection reagent.
In a laminar flow hood, to combine one microgram of plasmid DNA with a hundred microliters of MEM in a single tube at room temperature, be sure to use MEM that does not contain any supplements. Combine six microliters of lip with a hundred microliters of MEM in the other tube. The same amount can be used for double transfect, even though the lip DNA ratio will be halved in such cases.
To preserve the shelf life of the lip reagent, keep it refrigerated or in a benchtop cooler at all times, incubate the tubes for five minutes at room temperature. Transfer the mixture of DNA in MEM into the lipid tube and gently mix with APE Next incubate for 30 minutes at room temperature. After 25 minutes, add 60 microliters of 50 millimolar K neuronic acid to minimize cytotoxic damage to the cultured neurons.
Since we culture neurons on the underside of cover slips suspended above a GL monolayer, it is necessary to invert the cover slips and expose the neurons to the transfection solution. Carefully flip the cover, slips feet up using sterile forceps. Arrange them so they do not overlap.
Be careful not to scrape the neuron coated surface of the cover slip or the ggl coated surface of the dish. At the end of the 30 minute incubation, take approximately N 0.5 milliliters of medium from the six centimeter dish and gently mix with the DNA in the tube. Transfer the DNA solution back to the dish dripping evenly onto the surface of the medium.
Gently slide the dish back and forth in one direction, but be sure not to swell the medium. After mixing in one direction, stop and repeat in the perpendicular direction to evenly distribute the DNA lipectomy complexes throughout the dish. Incubate for 90 minutes in the tissue culture incubator at 37 degrees C and 5%carbon dioxide.
After incubation, flip the cover slips feet side down and arrange them so they do not overlap. Finally, allow expression of the DNA to proceed for the desired time, which is typically overnight to 48 hours in the incubator. Live imaging of GFP tagged dense core les in cultured hippocampal neurons requires a fluorescent microscope equipped with a CCD camera.
An imaging chamber with a temperature controller and an objective heater will be needed. The chamber should contain no open ports, a modification that can be included when ordering the chamber. Next, start up the microscope camera, fluorescent lamp and image acquisition software.
Turn on the power to the chamber platform and objective heaters. This is also a good time to apply a drop of oil to the objectives. Now prepare the heated platform and imaging chamber.
For ease of manipulation. Label one side of the chamber top with a permanent marker. Place the chamber top side up on the lid of a 50 milliliter tube, which is an ideal chamber holder.
Apply a ring of grease to the groove surrounding the hole in the side of the chamber labeled top. Avoid using the grease in excess since it will enter the chamber and reduce the observable area. Using forceps.
Attach a clean unused 18 centimeter cover slip to the top side of the chamber. Apply gentle pressure to the cover slip at its edges to create a tight seal within the recessed groove of the chamber. Turn the chamber over and apply a similar ring of grease to the groove on the opposite or bottom side of the chamber.
Transfer 750 microliters of imaging medium to the chamber. This is an excessive amount, but the grease should prevent the medium from spilling over and the excess will prevent bubbles from being trapped when the cell covered cover slip is applied. Keep the prepared chamber in the tissue culture incubator until needed.
Prolonged incubations such as an hour or more should be avoided as the imaging medium will evaporate and change in composition. For the final chamber assembly for lifestyle imaging, move the prepared chamber and a dish of transfected cover slips from the incubator to the tissue culture hood. Using sterile forceps, carefully remove one cover slip from the transfected dish.
Then touch the edge of the cover slip to a kim wipe to draw away the growth medium. Next, place the cover slip neuron side down onto the prepared chamber. Touch one side of the cover, slip to the grease first and apply pressure around the edges to bring the cover slip flat without trapping bubbles.
Excess imaging medium is expected to spill out, wipe off the excess imaging medium, but be careful not to slide the cover slip outta position. Move the chamber to the preheated platform heater and fasten the chamber in place. Next, transfer the chamber to the stage to reduce photobleaching and phototoxicity.
Adjust the lamp to the minimum amount of intensity necessary to find the transfected cells. Now find a cell of interest. Using a low magnification oil objective such as 40 x, it is beneficial to stick to a planned search pattern to ensure maximal cover slip coverage and to avoid redundant image acquisitions.
Once a desired field has been located, switch the channel of a fluorescently labeled protein of interest. Switch to a high magnification oil objective, such as 60 to a hundred x, and return the lamp intensity back to a high setting use stream acquisition or a comparable function of your imaging software to record the fluorescently labeled protein dynamics for fast moving organelles. The exposure time and delay between exposures should be minimized to produce recordings with a high frame rate.
Focal drift that is observed in the onscreen preview can be corrected in real time by manual focus adjustment. Be sure to take a phase image and any other accompanying images necessary for later analysis and presentation. Save all images before exploring the cover slip further and making the next recording.
Typically three to five recordings from one cover slip is considered successful and cover slips are imaged for 30 to 60 minutes each phototoxicity in general cell health should be kept in mind as transporters reduced in damaged cells. Live cell imaging observations typically consist of recordings, also known as stacks of images that show movement of fluorescently tagged organelles within the field of view. Here is a representative movie of dense core iCal transport.
The color has been inverted for easier observation. Notice the moving particles. CH graphs are images that illustrate particle movement by juxtaposing line scans where each time point is represented by a column in the graph.
This figure demonstrates how two particles moving in opposite directions are represented in a CH graph. Further analysis of chronographs can provide information about particle flux velocity, run length reversals, and stationary particles. Hi, we've just shown you how to acquire high speed recordings of dsco vesicle transport and cultured hippo capital neurons.
When doing this procedure, it is important to remember to preheat all components of the imaging chamber and platform to reduce stress in the cells during their transition from the incubator to the stage. You should work quickly but carefully. So that's it.
Thanks for watching and good luck with your experiments.
