Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

In immune cells, caspase-1, an inflammatory caspase, is a key immune-responsive component that exists in an inactive form. These inactive forms consist of a pro-domain fused with a catalytic domain and become active via proximity-induced dimerization.

To visualize caspase activation in vivo, begin with a culture of transfected human monocyte-derived macrophages expressing red fluorescent proteins and a pair of fluorescent complementation reporter proteins. The reporter proteins contain a pro-domain of caspase-1 fused with non-fluorescent fragments of the Venus protein, either Venus-N or Venus-C.

Treat the cells with an imaging medium containing lipopolysaccharides — a bacterial-derived inflammatory stimulant, and potassium ionophores — nigericin.

Lipopolysaccharides interact with pattern recognition receptors on the macrophage, initiating the assembly of an inflammasome complex.

Nigericin molecules enter the cell and bind to potassium ions, causing their efflux into the extracellular medium and decreasing the intracellular potassium ion concentration. This leads to the priming of inflammasome sensor proteins with adaptor proteins — a bridging protein between sensor protein and procaspase-1, forming the complex.

The pro-domains containing Venus fragments interact with the complex's adaptor proteins. This brings the two fragments in proximity, forcing them to refold and fluoresce.

Visualize the cells under an epifluorescence microscope.

The transfected cells appear red with green fluorescence spots, confirming caspase activation.

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 transfection parameters in the startup screen displayed. Press on Voltage, enter 1,000, and press Done to set it to 1000 volts. Press on Width, enter 40, and press Done to set the pulse to 40 milliseconds. Lastly, press on # Pulses, enter two, and press Done to set the number of electrical pulses to 2.

Take one of the electroporation tubes and fill it with three milliliters 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 re-suspension R-buffer for each 1 to 2 times 10 to the fifth cells, and mix gently with a P20 micropipette. Add 10 microliters of the cell suspension to each transfection tube, and mix gently with a P20 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 transfected 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 transfected 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 P1000 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 a confocal microscope, turn on the microscope and the fluorescent light source, following the manufacturer's 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 eyepiece, 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 of red cells that are also green. Count at least 100 red cells of minimum three fields.