Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

The overall goal of this model is to allow the simultaneous monitoring of multiple chemotaxis assays, or visualization of the multiple signalling events of a single chemotaxing cell. Overdeck's cell biologists have developed several different approaches to quantify chemotaxis behavior of the cells. Until recently, we are able to answer key questions in the chemotaxis field, such as, how to simultaneously monitor multiple chemotaxis assays.

The main advantage of this technology is to apply the principle of microfluidics to generate highly reproducible and reliable ingredients for multiple simultaneous chemotaxis assays with high resolution in real time. Demonstrating the procedure will be Dr.Xi Wen, a post doc from our laboratory. To assemble the holder, first use the levers to position the base in the vertical position so that the levers can tilt toward the user.

Then, briefly coat the surface of the 41 millimeter glass with 70%ethanol, and use a wipe to carefully remove the ethanol. Insert the glass into the holder base, and place a BSA coated 2 millimeter square cover slip on top of the glass. Next, insert the small O-ring into the bottom of the wafer housing, and mount the wafer housing into the holder base, with the elliptical hole at the rear of the instrument.

Pull the inner level of the holder base forward to the horizontal position, to lock the wafer housing in place. Use an air duster to blow any debris from the interior of the wafer housing. Then add 4 millileters of RPMI 1640 medium, supplemented with 0.1%BSA, to the housing.

Now, mount the BSA coated cell mobility analysis device chip into the center of the wafer housing with the structural face in contact with the glass, and with the positioning mark at the rear. Fit the two protrusions on the rubber gasket into the holes to affix the gasket to the bottom of the wafer clamp, and mount the large O-ring onto the top of the wafer housing. Then mount the wafer clamp with the sensor hole at the rear and pull the outer lever of the housing base forward to the horizontal position to lock the wafer clamp in place.

When the holder is assembled, place the bottom of the holder onto a light box to confirm that there are no air bubbles in the wells. Then remove the cover, transfer the assembly onto the plate on top of the cell mobility analysis device unit, and attach the sensor block to the holder. To perform the cell mobility assay, first we suspend differentiated HL60 cells in BSA supplemented RPMI 1640 medium, and a two times ten to the sixth cells per milliliter concentration.

Then, connect the cell mobility analysis device unit to the computer for the image acquisition, and turn on both devices. Next, open the cell mobility analysis device software. In the camera image control panel, use the horizontal and vertical lines to adjust the holder position in real time, and center the device in the camera image panel.

Then select Channel One to move the camera to the selected channel, and use Move Left of Move Right to adjust the X coordinate of the camera to center the view field horizontally. To vertically adjust the image to the center of the screen, turn the position knob on the front panel of the cell mobility analysis device. On the heater control panel, set the holder temperature to 37 degrees Celsius, and the plate temperature to 39 degrees Celsius.

To control the temperature using the thermal sensor connected to the holder, click Heat to start the heating, and then Holder. On the shooting panel, enter 15 seconds for the Interval and 30 minutes for the Time to set the intervals and the duration of the chemotaxis assay, respectively. For safety purposes, select the Heater off at the end of shooting box.

Then, save the file, input the specifics of the experiment into the memo panel, and confirm that the device has been centered in all of the channels. Now remove all of the buffer from the holder, and eight microliters of buffer from the third well from the top of the first channel. Using a syringe, inject two microliters of cells into the second well in the same channel, while monitoring the screen in real time to control the cell number and flow during the injection.

When the cells are aligned, immediately add back the eight microliters of buffer to the well, and repeat the cell injection in channels two through six, as just demonstrated. After all of the cells have been added, add two milliliters of buffer back to the holder, and add one microliter of the chemoattractant to the third well from the top in the appropriate channels. Finally, start the image acquisition.

In this representative experiment, the HL60 cells begin chemotaxing in a straight path immediately upon the injection of the chemoattractant, and continued for the entire 60 minutes of the assay, consistent with the gradient stability simulation results. Tracing the travel path and morphology of the cells allows the quantitative measurement and subsequent comparison of the chemotaxis behaviors, using a chemotaxis index that includes the total path length, directionality, speed, and roundness of the cells. Application of a fluorescent dye, in conjunction with the chemoattractant, allows the establishment of a linear relationship between the chemoattractant concentration, and the monitored flourescent dye intensity.

Further, in response to uniformly applied chemoattractant stimulation, the HL60 cells mediate a robust membrane translocation of GFP tact protein kinase D1.In a chemoattractant gradient, the HL60 cells actively recruit the kinase to the rear of the leading edge. Once mastered, this process can be completed in 30 minutes if performed properly. While attempting this procedure, it is important to strictly follow the manufacturer's instructions on the holder assembly, and monitor the cell injection.

After it's development, this technique paved the way for researchers in the field of chemotaxis to explore the possibility of multiple chemotaxis assays, such as a monogenism dictyostilium, or other types of mammallian cell systems. After watching this video, you should have a good understanding of how to assemble the unit, perform simultaneous chemotaxis assays, or simultaneously visualize multiple signalling events in chemotaxing cells.