This protocol is significant because it can be used to visualize and interrogate inflammatory caspase pathway at the molecular level in living cells in a particular disease state. The main advantage of this technique is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complexes in primary immune cells. With minor optimizations, this method can be adapted to other primary cells and a range of proteins activated by oligomerization, and its applicability is not limited to microscopic methodologies.
First, aspirate the media from fully differentiated macrophages on 10 centimeter dishes and wash the cell monolayer with warm serum free RPMI-1640 medium, removing all the media completely. Harvest the cells by adding two milliliters of 0.25%warm trypsin-EDTA solution per 10 centimeter dish. Gently pipette the trypsin-EDTA up and down over the entire dish with a P-1000 micropipette, then transfer the suspension to a 15 milliliter conical tube containing five milliliters of warm complete culture medium.
Check for cell detachment under a bright field microscope and detach again if needed. Aspirate the medium and resuspend the cells in 10 milliliters of 1X sterile PBS pre-warmed to 37 degrees Celsius. Then take a 20 microliter aliquot to determine the cell number using a hemocytometer.
Take one to two times 10 to the fifth cells per transfection and place them in a 15 milliliter tube. Bring to a final volume of 15 milliliters with pre-warmed 1X sterile PBS. Aspirate the PBS and centrifuge one more time for one minute at 250 x g.
Remove any residual PBS from the cell pellet using a P-200 micropipette. Take a 1.5 milliliter sterile microtube per transfection and add the appropriate reporter plasmid and caspase BiFC plasmids in the hood. Place the pipette station, device, tips, electroporation tubes, and pipette in a sterile laminar flow hood.
Connect and switch on the nucleofection device. Enter the transection parameters in the startup screen displayed. Press on Voltage, enter 1000, and press on Done to set it to 1000 volts.
Press on Width, enter 40, and press on Done to set the pulse to 40 milliseconds. Lastly, press on Pulses, enter 2, and press on Done to set the number of electrical pulses to 2. Take one of the electroporation tubes and fill it with three milliliter of electrolytic buffer E at room temperature.
Insert the electroporation tube into the pipette holder on the pipette station, ensuring that the electrode on the side of the tube is facing inwards and a click sound is heard when the tube is inserted. Take the cell pellet and add 10 microliters of pre-warmed resuspension R buffer for each one to two times 10 to the fifth cells, and mix gently with a P-20 micropipette. Add 10 microliters of the cell suspension to each transfection tube and mix gently with a P-20 micropipette.
Insert a tip into the pipette by pressing the push button to the second stop, ensuring that the clamp completely picks up the mounting stem of the piston in the tip. Next, press the push button on the pipette to the first stop and dip into the first tube containing cell or plasmid DNA mixture to aspirate the sample. Insert the pipette with the sample very carefully into the pipette holder, ensuring that the pipette clicks and is appropriately placed.
Press start on the touch screen and wait until the electric pulses are delivered. Slowly remove the pipette from the station and immediately add the transected cell suspension into the corresponding well with the pre-warmed antibiotic-free medium by slowly pressing the push button to the first stop. Gently rock the plate with transected cells and incubate for one to three hours in a humidified tissue culture incubator, then add 200 microliters of pre-warmed culture medium to each well.
Place the dish in the incubator and allow at least 24 hours for gene expression. The next day, inspect the cell viability and transfection efficiency using an epifluorescence microscope. Remove the media from the cells carefully with a P-1000 micropipette and add 500 microliters of the stimulus solution down the side of the well.
To run untreated control wells, add an imaging medium without the stimulus. To visualize the cells using an epifluorescence or confocal microscope, turn on the microscope and the fluorescent light source following the instructions. Select the 10 times or 20 times objective and place the culture dish on the microscope stage.
Find cells under the 568 nanometer filter with the eye piece. And focus on the cells expressing the reporter red cells. Count all the red cells in the visual field.
While in the same visual field, change to the 488 or 512 filters, count the number red cells that are also green. Count at least 100 red cells of minimum three fields. Calculate the percentage of Venus-positive transected cells per visual field and average the resulting percentages for each treatment well to get the standard deviation.
To image the cells using an epifluorescence or confocal microscope, visualize the live image of the cells on the computer screen as acquired by the camera. Fine tune the focus and position of the cells using the joystick control and focus wheel. Set the percentage laser power and exposure time for the 512 nanometer or 488 nanometer and 568 nanometer lasers, so that the signal in the image looks good without saturation.
Turn on the live capture and examine the resulting image, ensuring that a distinct peak is seen for each floor in the display histograms for both channels. While visualizing the live image of the cells, take multiple representative images of a field that contains one or more cells expressing the mCherry or dsRedmito reporter for each well of the plate. Selected CD-14 positive monocytes incubated with GM-CSF for seven days showed changes in the cell morphology over the course of the differentiation period, going from spherical suspension cells to spindly and fully attached, and lastly, to more spread cells when fully differentiated.
Fully differentiated cells were transected with the caspase BiFC pairs VC and VN along with the reporter plasmid dsRedmito, which was used to label the transected cells. Untreated cells showed no Venus fluorescence. And in nigericin treated cells, caspase-1 BiFC appeared as a single punctum with the typical shape of ASC specks.
Quantitation of Venus-positive MDM showed that the highest percentage of caspase-1 BiFC is seen in the LPS plus nigericin treatment group. On plasmid titration of caspase-1, 4, and 5 pro-BiFC pairs, the highest percentage of Venus-positive cells resulted from 400, 500, and 1, 000 nanograms of transected plasmid, respectively. Confocal images obtained with a 20 times objective of a field of cells 24 hours post-transection showed that the untreated transected cells are viable as mitochondria are intact.
After treatment with heme, the caspase-1 complex appears as a single green punctum similar to the one induced by nigericin. Remember to avoid harsh conditions and excessive manipulation of the cells during differentiation, transfection, and treatment as it can result in a spontaneous activation and high background levels of caspase activation.
