Name: imaging g protein-coupled receptor-mediated chemotaxis and its signaling events in neutrophil-like hl60 cells
Uploaded: 2016-09-14T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells at JoVE.com

This work is supported by the intramural fund of NIAID, NIH.

1. Culture and Differentiation of Human Neutrophil-like HL60 Cells
<ol>
	<li>Prepare RPMI (Roswell Park Memorial Institute) 1640 culture medium which contains RPMI 1640 medium, 10 % (v/v) fetal bovine serum (FBS), 0.1 mM sodium pyruvate, and 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Warm the RPMI 1640 culture medium to 37 &#176;C.</li>
	<li>Using a hemocytometer determine the cell density of HL60 cells to make sure the cells are in the log-phase growth stage. 
	NOTE: Cells are usually in log phase on the third day after passaging. The density of log-phase cells is usually 6-8 x 105 cells/ml. The cell density required to start a new culture is about 1.5 x 105 cells/ml. 
	NOTE: For example, for a 10 ml new culture in a T-75 flat flask, if the cell density of log-phase cells is 6 x 105 cells/ml, then 2.5 ml of log-phase cells (6 x 105) is diluted to 1.5 x 105 cells/ml in 10 ml new culture. To make a final volume of 10 ml culture, 7.5 ml of RPMI 1640 culture medium is added.</li>
	<li>Place the calculated volume of warm RPMI 1640 culture medium into a flat flask. For a T-75 flat flask culture, the total volume of the cell culture is 10 ml.</li>
	<li>Add the calculated volume of log-phase growing HL60 cells into the flat flask to reach a cell density of 1.5 x 105 cells/ml.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified incubator with 5% CO2. 
	NOTE: The doubling time of HL60 is almost 24 hr.</li>
	<li>Passage the cells with RPMI 1640 culture medium every 2-3 days. It is important that the cell density of the HL60 cells never exceeds 1.5 x 106 cells/ml and the cell passage lasts no longer than 2 months.</li>
	<li>Differentiate the HL60 cells 5 days before the experiments. 
	NOTE: RPMI 1640 differentiation medium contains RPMI 1640 medium, 10% (v/v) fetal bovine serum (FBS), 0.1 mM sodium pyruvate, 25 mM HEPES, and 1.3% dimethyl sulfoxide (DMSO). In other words, it is 1.3% DMSO in RPMI 1640 culture medium.
	<ol>
		<li>To differentiate HL60 cells, pre-warm the RPMI 1640 culture medium to 37 &#176;C. Add the warm RPMI 1640 culture medium into a flat flask.</li>
		<li>For a final 10 ml HL60 differentiation culture, add 130 ul DMSO directly to the RPMI 1640 culture medium in the flat flask while swirling the flask so that the DMSO is rapidly diluted by the RPMI 1640 culture medium.</li>
		<li>Add log-phase HL60 cells to a final cell density of 1.5 x 105 cells/ml in a final 10 ml RPMI 1640 differentiation medium. Culture the HL60 cells in RPMI 1640 differentiation medium at 37 &#176;C in a humidified incubator with 5 % CO2 for 5 days.</li>
	</ol>
	</li>
</ol>
2. Coating the Cover Glass Surface of the 4well Chamber
<ol>
	<li>Take the bottle of 2% gelatin stock solution from the refrigerator, clean it with 70% ethanol, and place it in the hood. Take 1 ml 2% gelatin stock solution and return the remaining gelatin stock solution to the refrigerator immediately.</li>
	<li>Allow the gelatin solution to completely liquefy at 37 &#176;C and pipette it thoroughly. Dilute the 2% gelatin stock solution with 9 ml pre-warmed 1x Hanks&#39; balanced salt solution (HBSS) buffer to a final concentration of 0.2% gelatin.</li>
	<li>Add 0.5 ml or 2 ml 0.2% gelatin in HBSS to the two middle wells of a 4-well chamber or a 1-well chamber with a cover glass in the bottom, respectively. Tilt the plates in several directions so that the liquid covers the entire surface area.</li>
	<li>Place the plates in a 37 &#176;C incubator. The plates will be ready for use in 1 hr. They can be used for up to 5 days. Just before seeding cells, remove the gelatin solution from the 4-well or 1-well chambers.</li>
</ol>
3. Chemotaxis Assay Using Cell Mobility Analysis Device
<ol>
	<li>Prepare and warm RPMI 1640 starving medium, which is RPMI medium containing 0.1 mM sodium pyruvate (optional) and 25 mM HEPES.</li>
	<li>Prepare 100 &#956;l of 100 nM fMLP (N-formylmethionyl-leucyl-phenylalanine) in RPMI 1640 starving medium.</li>
	<li>Prepare 3 ml of 1% bovine serum albumin (BSA)/RPMI 1640 medium which contains 1% BSA in RPMI 1640 starving medium and 10 ml of 0.1% BSA/RPMI 1640 medium.</li>
	<li>Coat a 22 mm square coverslip and a 5 &#956;m cell mobility analysis device chip with freshly prepared 1% BSA/RPMI 1640 medium for 1 hr at RT.</li>
	<li>Assemble the Holder Using the Following Procedure:
	<ol>
		<li>Position the holder base with the levers in the vertical position so that the levers can tilt toward the user. Apply 70% ethanol solution to the surface of the 41 mm glass. Carefully wipe the surfaces with a wipe, taking care not to scratch them.</li>
		<li>Insert the 41 mm glass into the holder base. Put the BSA-coated 22 mm square coverslip on top of the 41 mm glass in the holder base. 
		NOTE: The coated square coverslip between the 41 mm glass and the chip allows chemotaxis to occur on the substratum of the coating surface.</li>
		<li>Insert the O-ring (small) into the bottom of the wafer housing, and mount the wafer housing into the holder base with the elliptical hole at the rear. Pull the inner level of the holder base forward to the horizontal position to lock the wafer housing in position.</li>
		<li>Blow any dust from the interior of the wafer housing with an air duster. Add 4 ml of 0.1% BSA/RPMI 1640 medium into the wafer housing.</li>
		<li>Mount the BSA-coated cell mobility analysis device chip in the center of the wafer housing with the structural face downwards in contact with the glass. 
		NOTE: That the chip needs to be inserted with the positioning mark (the hole in the top of the chip) at the rear.</li>
		<li>Affix the rubber gasket to the bottom of the wafer clamp by fitting the two protrusions on the rubber gasket into the holes.</li>
		<li>Mount the O-ring (large) onto the top of the wafer housing. Mount the wafer clamp with the sensor hole at the rear. Pull the outer lever of the housing base forward to the horizontal position to lock the wafer clamp in place.</li>
		<li>After assembling the holder, place the bottom of the holder on the light box to confirm that there are no air bubbles in the wells. If air bubbles are detected, remove them using the provided plastic syringe fitted with a sample loading tip.</li>
		<li>Remove the cover and put the assembly onto the plate on top of the cell mobility analysis device unit. Attach the sensor block to the holder.</li>
	</ol>
	</li>
	<li>Prepare HL60 Cells for Chemotaxis Assay:
	<ol>
		<li>Calculate the cell density of the differentiated HL60 cells with a hemocytometer. After 5 days of differentiation, the cell density is around 1.0 x 106 cells/ml.</li>
		<li>Calculate the final volume of HL60 cells at a final cell density of 2 x 106 cells/ml. For example, if the cell density of the differentiated HL60 cells is 1.0 x 106 cells/ml, then 10 ml of differentiated HL60 cells will be resuspended with 5 ml of 0.1% BSA/RPMI 1640 medium to reach 2 x 106 cells/ml.</li>
		<li>Centrifuge differentiated HL60 cells at 200 x g for 5 min at RT. Remove the supernatant and resuspend the cells with the calculated volume of 0.1% BSA/RPMI 1640 medium at step 3.6.2) to a final cell density of 2 x 106 cells/ml.</li>
	</ol>
	</li>
	<li>Start up the cell mobility analysis device unit and PC for image acquisition. Connect the unit to the PC. Turn on the cell mobility analysis device. Turn on the PC.</li>
	<li>Start Up the Cell Mobility Analysis Device Software.
	<ol>
		<li>Observe 5 control panels: camera, heater, shooting, memo, and camera images.</li>
		<li>On the camera image control panel, use &#34;Horizontal line&#34; or &#34;Vertical line&#34; to adjust the holder/view position in real time to center the camera in the camera image panel.</li>
		<li>On the camera control panel, select CH1 to CH6 to move the camera to the defined channel. To center the view fields of a channel, click &#34;Move L&#34; or &#34;Move R&#34; to adjust the X-coordinate of the camera and center the camera&#39;s position horizontally.</li>
		<li>Adjust the Y-coordinate of the camera by turning the position knob on the front panel of the cell mobility analysis device unit to vertically adjust the image to center the screen.</li>
		<li>On the heater control panel, set the &#34;holder temp&#34; to 37.0 &#176;C and the &#34;plate temp&#34; to 39 &#176;C. Click &#34;Heat&#34; to start heating. Also click &#34;holder&#34; to control the temperature using the thermal sensor connected to the holder.</li>
		<li>On the shooting panel, enter &#34;15 sec&#34; at &#34;Interval&#34; and &#34;30 min&#34; at &#34;Time&#34; for the intervals and the duration of the chemotaxis assay. Check &#34;Heater off at the end of shooting&#34; for safety reasons. Designate a destination for the stored images.</li>
		<li>On the memo panel, input the specifics of the experiments, such as cell line used, whether or not treatment was applied, and the concentration of chemo-attractants applied.</li>
		<li>Confirm the center position of all of the channels that will be used in the experiments, and repeat the adjustment for all channels if needed.</li>
	</ol>
	</li>
	<li>Inject and Align Cells as Follows:
	<ol>
		<li>Remove the buffer from the holder, and take out 8 &#956;l of buffer from the third well from the top for each channel.</li>
		<li>Use a syringe to inject 2 &#181;l of cells into the second well in the same channel while monitoring the screen in real time to control cell number and flow during injection. After the cells are nicely aligned, immediately add the 8&#160;&#181;l of buffer back. Repeat the same procedure for the other channels (Figure 1B).</li>
	</ol>
	</li>
	<li>Add 2 ml buffer back to the holder, and add 1 &#956;l 100 nM fMLP to the third well from the top. Start image acquisition and save the data. Analyze the data obtained using image tracing software9 (Figure 2).</li>
</ol>
4. Transfection with Electroporation
<ol>
	<li>Differentiate HL60 cells 5 days before the experiment as described in Section 1. Coat 4-well or 1-well chambers as instructed in Section 2.</li>
	<li>Prepare and warm RPMI 1640 electroporation recovery medium, which contains RPMI 1640 medium, 20% (v/v) FBS, 0.1 mM sodium pyruvate, and 25 mM HEPES, to 37 &#176;C.</li>
	<li>Right before starting the transfection experiment, add either 300 &#956;l or 3 ml RPMI 1640 culture medium into the wells of the pre-coated 4-well or 1-well chambers, respectively.</li>
	<li>Incubate the chambers in a humidified 37 &#176;C incubator with 5 % CO2. Use these chambers immediately or store in the incubator for future use (two weeks).</li>
	<li>Count the cell density of the differentiated HL60 cells with a hemocytometer. 
	NOTE: The cell density often reaches about 1.0 x 106 cells/ml. Cell density greater than 3.0 x 106 cells/ml usually gives undesirable transfection efficiency. As HL60 cells are suspension cells, no resuspension is required.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at RT. Remove the supernatant and resuspend 2 x 106 HL60 cells in 100 &#956;l of transfection reagent.</li>
	<li>Add 4 &#956;g of GFP-PKD1 plasmid9 into the 100 &#956;l mixture of cells and transfection reagents and mix gently and thoroughly. Add the mixture into the cuvette provided.</li>
	<li>Select the manufacturer&#39;s pre-set program for the human leukocyte cell line and perform the electroporation. 
	NOTE: Further information is available from the manufacturer&#39;s website.</li>
	<li>After electroporation, immediately and gently add pre-warmed 500 &#956;l RPMI 6140 electroporation recovery medium to the cells, and gently transfer the cells from the cuvette to a 1.6ml tube with the plastic pipettes provided.</li>
	<li>Incubate the cells for 30 min in a humidified 37 &#176;C incubator with 5% CO2. Seed 100 &#956;l or 500 &#956;l of cells into one well of a 4-well or a 1-well chamber, respectively.</li>
	<li>Add RPMI 1640 culture medium to a final volume of 1 ml or 4 ml to that same well of a 4-well or a 1-well chamber, respectively. Incubate the cells in a humidified 37 &#176;C incubator with 5 % CO2 for 3 hr. Pre-warm RPMI 1640 starving medium to 37 &#176;C.</li>
	<li>Using a 1ml pipette, gently remove 0.8 ml or 3 ml of RPMI 1640 culture medium from the well of a 4-well chamber or a 1-well chamber, respectively. Be gentle and avoid sucking away adhering HL60 cells. 
	NOTE: HL60 cells grow and are differentiated in suspension media. After being differentiated or with certain treatments, HL60 cells adhere to the substratum coated with polylysine, collagen, gelatin, or fibronectin.</li>
	<li>Add 0.8 ml or 3 ml of RPMI 1640 starving medium into the well of a 4-well or 1-well chamber, respectively. Wait 1 hr. 
	NOTE: At this point the cells are ready for imaging experiments9.</li>
</ol>
5. Monitoring GPCR-mediated Membrane Translocation of PKD1 by Multi-channel Fluorescent Microscopy
<ol>
	<li>Prepare a fresh mixture of 100 nM fMLP and 1 &#956;g/ml Alexa 594 in RPMI 1640 starving medium as stimuli. 
	NOTE: The mixture of fMLP and Alexa 594 needs to be freshly made to ensure maximum efficacy.</li>
	<li>Mount one 4-well chamber seeded with cells over a 40X oil lens.</li>
	<li>Set the Fluorescent Microscope in the Multichannel Configuration as Follows:
	<ol>
		<li>Set the green emission channel (500-530 nm) for GFP-tagged PKD1, red emission channel (580-620 nm) for chemoattractant fMLP (Alexa 594), and transmitted light channel for identifying adhering HL60 cells.</li>
		<li>Start &#34;live&#34; acquisition and optimize the acquisition conditions of the three channels:</li>
		<li>Fine-tune between intensity and laser power for all three channels to minimize photobleach for a longer period of imaging.</li>
		<li>If necessary, average every 2 to 4 frames to decrease the background noise ratio for better quality of images. Use a 1 sec interval.</li>
	</ol>
	</li>
	<li>Search for adhering cells that express strong PKD1-GFP in the view of the GFP and DIC (differential interference contrast) channel. Adhering cells are morphologically flat and do not move rapidly.</li>
	<li>After identifying adhering HL60 cells that highly express GFP-PKD1, optimize the acquisition condition by lowering the laser power while increasing the detector gain for each channel to decrease the photobleach for long-term imaging (Figure 3A).</li>
	<li>Take up 100 &#956;l of fMLP/Alexa 594 solution with a 200 &#956;l pipetter and set aside. Select the &#34;time-lapse acquisition&#34; and set the intervals to 2 sec and the number of frames to acquire to 100. 
	NOTE: the intervals and number of frames to acquire depend on the purpose of experiments. 1-2 sec intervals and 100 frames are often used for imaging experiments, as in Figure 3. For slowly migrating cells, longer intervals are necessary for long-term imaging. In contrast, for rapidly migrating cells, shorter intervals are required for observing the continuous details of the cell migration.</li>
	<li>Carefully take off the lid of a 4-well chamber so that the position of the chamber is not changed. Start acquisition immediately and acquire 3-5 images of the unstimulated cells while positioning the 200 &#956;l pipetter directly over the cells using the spot of laser light as a guide.</li>
	<li>Pipette the fMLP/Alexa 594 mixture over the cells being imaged with a quick and even flow and finish the complete set of time-lapse acquisition. Name and save the data that will be subjected to further data analysis.</li>
</ol>
6. Imaging Chemotaxing Cells in Visible and Controllable Chemo-attractant Stimuli
<ol>
	<li>Using a 10 &#956;l micropipetter and injection tips, backfill a micropipette with 30 &#956;l freshly prepared mixture of fMLP and Alexa594.</li>
	<li>Attach the micropipette to a micropipette holder mounted on top of a microscope stage and connect the tubing to the pressure supply unit, an apparatus that provides a constant pressure to release fMLP/Alexa594 solution from the micropipette to generate a steady gradient.</li>
	<li>Turn on the pressure supply and set the compensation pressure (Pc) to 70 hPa.</li>
	<li>Mount a 1-well chambered coverslip seeded with HL60 cells expressing PKD1-GFP over a 40X oil lens on a confocal microscope or its equivalent fluorescent microscope.</li>
	<li>Set acquisition in channel mode in the following configuration: green channel (500-530 nm) for GFP-tagged PKD1; red channel (580-620 nm) for chemoattractant fMLP (Alexa594); and transmitted light channel for identifying adhering HL60 cells.</li>
	<li>Start &#34;live&#34; acquisition and optimize the acquisition conditions for the three channels: adjust the intensities of each channel for quantitative analysis of imaging data; fine-tune between intensity and laser power for all three channels to obtain images with the best quality and minimize the photo-bleach. If necessary, average every 2 to 4 frames for better image quality. We usually use a 1 sec interval.</li>
	<li>Start &#34;live&#34; acquisition and search for adhering cells that express strong PKD1-GFP in the view of the GFP and transmitted light channels. Using bright-field optics, center the micropipette in the center field of view (Figure 4A) in order to visualize the fMLP gradient and the adhering HL60 cells expressing PKD1-GFP.</li>
	<li>Select time-lapse acquisition and set the intervals to 2 sec intervals and the number of frames to acquire to 100. 
	NOTE: After collecting each time-lapse series, name and save the data that will be subjected to further data analysis.</li>
</ol>

<ol>
	<li>Pocha, S. M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+mechanisms+of+single+and+collective+cell+migrations+in+Drosophila:+themes+and+variations.">Cellular and molecular mechanisms of single and collective cell migrations in Drosophila: themes and variations.</a> Annual review of genetics. 48, 295-318 (2014).</li><li>Wen, Z., Zheng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+guidance+of+nerve+growth+cones.">Directional guidance of nerve growth cones.</a> Current opinion in neurobiology. 16, 52-58 (2006).</li><li>Bravo-Cordero, J. J., Hodgson, L., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+cell+invasion+and+migration+during+metastasis.">Directed cell invasion and migration during metastasis.</a> Current opinion in cell biology. 24, 277-283 (2012).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+polymorphonuclear+leukocytes.">Chemotaxis by polymorphonuclear leukocytes.</a> The Journal of cell biology. 77, 269-287 (1978).</li><li>Jin, T., Xu, X., Hereld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis,+receptors+and+human+disease.">Chemotaxis, receptors and human disease.</a> Cytokine. 44, 1-8 (2008).</li><li>Dong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P-Rex1+is+a+primary+Rac2+guanine+nucleotide+exchange+factor+in+mouse+neutrophils.">P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils.</a> Current biology : CB. 15, 1874-1879 (2005).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+sensing+requires+G+beta+gamma-mediated+PAK1+and+PIX+alpha-dependent+activation+of+Cdc42.">Directional sensing requires G beta gamma-mediated PAK1 and PIX alpha-dependent activation of Cdc42.</a> Cell. 114, 215-227 (2003).</li><li>Van Haastert, P. J., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis:+signalling+the+way+forward.">Chemotaxis: signalling the way forward.</a> Nature reviews: Molecular cell biology. 5, 626-634 (2004).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR-Mediated+PLCbetagamma/PKCbeta/PKD+Signaling+Pathway+Regulates+the+Cofilin+Phosphatase+Slingshot+2+in+Neutrophil+Chemotaxis.">GPCR-Mediated PLCbetagamma/PKCbeta/PKD Signaling Pathway Regulates the Cofilin Phosphatase Slingshot 2 in Neutrophil Chemotaxis.</a> Mol Biol Cell. , (2015).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> The Journal of experimental medicine. 115, 453-466 (1962).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> The Journal of cell biology. 75, 606-616 (1977).</li><li>Zicha, D., Dunn, G. A., Brown, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> Journal of cell science. 99 (Pt4), 769-775 (1991).</li><li>Muinonen-Martin, A. J., Veltman, D. M., Kalna, G., Insall, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+chamber+for+direct+visualisation+of+chemotaxis.">An improved chamber for direct visualisation of chemotaxis.</a> PloS one. 5, e15309 (2010).</li><li>Bozzaro, S., Fisher, P. R., Loomis, W., Satir, P., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guenther+Gerisch+and+Dictyostelium,+the+microbial+model+for+ameboid+motility+and+multicellular+morphogenesis.">Guenther Gerisch and Dictyostelium, the microbial model for ameboid motility and multicellular morphogenesis.</a> Trends in cell biology. 14, 585-588 (2004).</li><li>Xu, X., Jin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+G-protein+coupled+receptor+(GPCR)-mediated+signaling+events+that+control+chemotaxis+of+Dictyostelium+discoideum.">Imaging G-protein coupled receptor (GPCR)-mediated signaling events that control chemotaxis of Dictyostelium discoideum.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Lin, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dynamic+temporal+and+spatial+concentration+gradients+using+microfluidic+devices.">Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.</a> Lab Chip. 4, 164-167 (2004).</li><li>Eiseler, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D1+regulates+cofilin-mediated+F-actin+reorganization+and+cell+motility+through+slingshot.">Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot.</a> Nature cell biology. 11, 545-556 (2009).</li><li>Roddie, P. H., Paterson, T., Turner, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+primary+acute+myeloid+leukaemia+blasts+and+myeloid+leukaemia+cell+lines.">Gene transfer to primary acute myeloid leukaemia blasts and myeloid leukaemia cell lines.</a> Cytokines Cell Mol Ther. 6, 127-134 (2000).</li><li>Schakowski, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+non-viral+method+for+transfection+of+primary+leukemia+cells+and+cell+lines.">Novel non-viral method for transfection of primary leukemia cells and cell lines.</a> Genet Vaccines Ther. 2, 1 (2004).</li><li>Uchida, E., Mizuguchi, H., Ishii-Watabe, A., Hayakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+efficiency+and+safety+of+non-viral+vector-mediated+gene+transfer+into+a+wide+range+of+human+cells.">Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells.</a> Biol Pharm Bull. 25, 891-897 (2002).</li></ol>

The authors declare no competing financial interest for this work.

In the present study, we show two examples of chemotaxis assays: first, simultaneous monitoring of multiple chemotaxis assays by the cell mobility analysis device; and second, visualization of the chemoattractant gradient and the spatiotemporal dynamics of signaling events in the same cells in real time.
Cell mobility analysis device for multiple simultaneous chemotaxis assays
In the present study, we introduced a detailed protocol to perform multiple simultaneous chemotaxis assays using a cell mobility analysis device. This device allows user to observe cellular chemotaxis behavior with a 10X objective lens in conventional bright-field observation. Because of the diffusion-dominant characteristics of the fluid flow, the cell mobility analysis device generates highly predictable, stable gradients and allows up to six chemotaxis assays to be carried out simultaneously. The critical steps of the protocol are to obtain reliable gradients and to align the cells on the terrace line. The user should strictly follow the manufacturer&#39;s instructions for holder assembly and injection of chemoattractants and cells. Detailed instructions are also available online. Compared to alternative chemotaxis methods11-13,15, this device significantly improves the reliability and efficiency of chemotaxis assays. Four sizes of cell mobility analysis device chip in sizes of 4, 5, 6, and 8 &#956;m are available to accommodate different types and sizes of cells. We found that a 4 or 5 &#956;m cell mobility analysis device chip is suitable for the HL60 and D. discoideum cells, which are about 10-15 &#956;m in diameter. However, one limitation is that this device is not suitable for all types of cells. We had little success using the cell mobility analysis device chemotaxis assay with Raw267.4 cells. The reason may be that Raw267.4 cells migrate too slowly. The time required for efficient chemotaxis of Raw267.4 cells might be much longer than the time a gradient is maintained by the device. Instead, a transwell migration assay worked well for Raw267.4 cells9. Another limitation is that fluorescent observation is not possible with the current device. A future direction is to monitor fluorescent imaging with higher magnification. This is possible with the improved cell mobility analysis device, which is equipped with fluorescent detection and a 100X objective lens. In addition, the number of simultaneous assays is also increased to 12. All these improvements facilitate the enhanced throughput of chemotaxis assays and the observation of subcellular dynamics in migrating cells.
High transfection efficiency of HL60 cells to express fluorescent protein-tagged protein
HL60 cells are an actively dividing leukemia cell line and grow in suspension. As previously reported18-20, HL60 cells are resistant to gene transfer. Both lipid and electroporation gene transfer have been tested, and higher transfection efficiency was obtained with electroporation, as described in detail in section 4. Obtaining high viability after electroporation is critical for obtaining high transfection efficiency because of the severe damage that results from electroporation. Subsequently, thorough and gentle cell handling are required, especially after electroporation. All media have to be pre-warmed and gently added to the cells in any steps after electroporation. It is also critical to minimize the exposure time of cells to the electroporation reagent. To avoid cell death, after electroporation, RPMI 1640 electroporation recovery medium must be added to the cells immediately. We found that 20% FBS in the recovery medium gives a much higher cell recovery rate than 10% FBS. After electroporation, incubation in RPMI 1640 electroporation recovery medium for 30 min is critical for greater viability and transfection efficiency. To further increase transfection efficiency, we also used 4 &#956;g plasmid DNA per transfection, which is almost the twice amount of plasmid recommended by the manufacturer.
There are two major limitations on electroporation transfection: the transiency of the protein expression and the cell number limit (2 x 106 cells per transfection). In undifferentiated cells, expression is detectable only during the first couple of cell divisions, since the vector plasmid is diluted by half after each cell division. For The differentiated HL60 cells survive no more than 48 hr. As a result, any experiment requires fresh transfections 6 hr prior to the experiment. The cell number for one electroporation merely meets the minimum requirement for any biochemical assays. For the purpose of repetitive use or large quantity, it is strongly recommended that an undifferentiated HL60 cell line be established that stably expresses the protein of interest which has been tagged with a fluorescent protein by a viral vector, if a viral vector is available or can be constructed.

Eukaryotic cells sense and move toward the higher concentration within a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis1, neuron patterning2, metastasis of cancer cells3, recruitment of neutrophils to sites of inflammation4, and the development of the model organism Dictyostelium discoideum5. In general, eukaryotic cells sense chemoattractants using G protein-coupled receptors5. The engagement of chemoattractants with these receptors promotes dissociation of the heterotrimeric G proteins G&#945; and G&#946;&#947;, which in turn activate downstream signal transduction pathways that ultimately regulate the spatiotemporal organization of the actin cytoskeleton to drive cell migration5-9.
Cell biologists have been developing and improving chemotaxis assays to examine how G protein-coupled receptor (GPCR) signaling mediates directed cell migration. The Boyden chamber or transwell migration assay was developed in 1960 by Boyden10. The assay works by creating a gradient of chemoattractant compounds between two wells that are separated by a microporous membrane. Its simplicity and ease of use make it the most widely used chemotaxis assay to date. However, the assay does not enable the migrating process of the cells to be visualized. The Zigmond chamber is the first visual microfluidic device that allows clear imaging of cell migration on a coverslip across a narrow constriction toward a source chemoattractant11. Dunn12 and Insall13 modified and improved the high-resolution and long-term imaging capability of the Zigmond chamber chemotaxis assay. Because of the highly predictable, diffusion-dominant characteristics of fluid flow, microfluidics has been providing solutions for next-generation chemotaxis assays such as EZ-TAXIScan (a cell mobility analysis device).
With the stability of the gradient ensured, the device allows six chemotaxis assays to be carried out simultaneously (Figure 1A). In contrast to the directionally fixed gradients generated in the various chamber assays above, the needle or micropipette assay developed by Guenther Gerisch generates a gradient with a movable source14. In the assay, chemoattractant is released from a movable micropipette to generate a stable gradient. With this needle assay, researchers found that different cells generate pseudopods with fundamentally different characteristics. Applying fluorescent microscopy, we were able to visualize the gradient to facilitate its quantitative measurement throughout15. In this study, we describe detailed methods for preparing chemotactic HL60 (human promyelocytic leukemia) cells, simultaneously monitoring multiple chemotaxis assays with the cell mobility analysis device, and visualizing the GPCR-mediated spatiotemporal dynamics of signaling molecules such as protein kinase D1 in single live cells in response to visible, spatiotemporally controllable chemoattractant stimuli. Our advanced imaging methods can be applied to general chemotaxis studies, and are especially suitable for mammalian cell systems.

<table><tbody><tr><td>RPMI 1640 Medium GlutaMAX</td><td>Life technologies</td><td>61870-036</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher Scietific</td><td>11360-070</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>1 M HEPES sterile solution, pH 7.3</td><td>Quality Biological Inc.</td><td>A611-J848-06</td><td></td></tr><tr><td>Penicillin streptomycin solution</td><td>Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Nucleofector&lt;sup&gt;TM&lt;/sup&gt; 2b</td><td>Lonza</td><td>AAB-1001</td><td></td></tr><tr><td>Amaxa&lt;sup&gt;TM&lt;/sup&gt; Cell Line Nucleofector&lt;sup&gt;TM&lt;/sup&gt; Kit V including Nucleofector&lt;sup&gt;TM&lt;/sup&gt; Solution, Singe use pipettes, Amaxa&lt;sup&gt;TM&lt;/sup&gt; certified 100 ml aluminum electrode cuvettes</td><td>Lonza</td><td>VCA-1003</td><td></td></tr><tr><td>Lab-Tek chambered #1.0 Borosilicate Coverglass</td><td>Nalge Nunc International Inc</td><td>155383</td><td></td></tr><tr><td>2% Gelatin solution</td><td>Sigma-Aldrich</td><td>G1393</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>HBSS (Hanks&amp;rsquo; Balanced Salt Solution)</td><td>Life technologies</td><td>14025-076</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A3803</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Single well Lab-Tek II coverglass chambers</td><td>Nalge Nunc International Inc</td><td>155361</td><td></td></tr><tr><td>Four-well Lab-Tek II coverglass chambers</td><td>Nalge Nunc International Inc</td><td>155383</td><td></td></tr><tr><td>Alexa 594</td><td>Thermo Fisher Scientific</td><td>A-10438</td><td></td></tr><tr><td>fMLP</td><td>Sigma -Aldrich</td><td>F3506-5MG</td><td></td></tr><tr><td>Cover glass thickness (2) 22 mm x 22 mm</td><td>Corning</td><td>2855-22</td><td></td></tr><tr><td>EZ-TAXIScan</td><td>Effector Cell Institute, Inc.</td><td>MIC-1001</td><td></td></tr><tr><td>EZ-TAXIScan chip (5 mm)</td><td>Effector Cell Institute, Inc.</td><td>EZT-F01-5</td><td></td></tr><tr><td>1701RN 10 &amp;mu;l syringe</td><td>Hamilton</td><td>80030</td><td></td></tr><tr><td>Femtotips II Injection tips</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Femtotips II</td><td>Eppendorf</td><td>930000043</td><td></td></tr><tr><td>TransferMan NK2, including motor module, X head with angle adjuster, and Positioning aids.&amp;nbsp;</td><td>Eppendorf</td><td>5188900056</td><td></td></tr><tr><td>DIAS software</td><td>Solltech Inc.</td><td></td><td></td></tr><tr><td>LSM 780 META or equivalent confocal microscope with a 40X 1.3 NA or 60X 1.4 NA oil DIC Plan-Neofluar objective lens</td><td>Carl Zeiss</td><td></td><td></td></tr></tbody></table>

imaging g protein-coupled receptor-mediated chemotaxis and its signaling events in neutrophil-like hl60 cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. 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.

Simultaneous imaging of chemotaxis of multiple HL60 cells using cell mobility analysis device
Based on the principle of microfluidics16, the manufacturer has provided simulated profiles of gradients: a gradient is generated within 1 min, stabilized within 5 min, and maintained over 2 hr. The highly predictable profiles of the stable gradients generated by microfluidics allow multiple chemotaxis assays to be carried out simultaneously. In the present study, we observed three simultaneous chemotaxis assays (Figure 2A and Movie 1). We found that HL60 cells started chemotaxing immediately after the chemoattractant was injected into the well of the chemoattractant, and kept chemotaxing in a straight path for the following 60 min, consistent with the simulation results for gradient stability. Tracing the travel path and morphology of the cells allows quantitative measurements and subsequent comparison of the chemotaxis behaviors using a chemotaxis index that includes total path length, speed, directionality, and roundness of the cells (Figure 2B). Total path length is the sum of the lengths of the line segments connecting the centroids of the path. Speed is obtained by dividing the total path length by the time. Directionality is measured upward and is defined as: (Y coordinate of the end of the path minus Y coordinate of the beginning) divided by total path length. This gives 1.0 for an object moving directly upward. The roundness of the cell is a measure (in percent) of how efficiently a given amount of perimeter encloses area. A circle has the largest area for any given perimeter and has a roundness parameter of 100%. A straight line encloses no area and has a roundness parameter of 0%. We show the quantitatively measured chemotaxis behavior as described by the selected chemotaxis parameters (Figure 2C).
Monitoring PKD subcellular localization in HL60 cells under a spatiotemporally visible and controllable fMLP stimulus
It is a great technical advance to apply fluorescently labeled and controllable chemoattractant stimulation to an experimental system. Historically, we have applied either homogeneous (also called uniform) stimulation or gradient stimulation to observe cell response and behaviors. However, &#34;blind&#34; stimulation not only provides no spatiotemporal information on how the stimulus reaches the cells, but also casts doubt on any &#34;abnormal&#34; observations of cell response to stimulation, simply because we do not see the stimulus. We have previously shown that fluorescent dye (Alexa594) can be applied with chemoattractant to establish a linear relationship between chemoattractant concentration and monitored fluorescent dye intensity15. With an acquisition configuration of green fluorescent protein (GFP), a red emission of fluorescent dye (Alexa594), and transmitted light, we are able to monitor the adhering cells, the application of the stimulus, and the cell response to the stimulus (Figure 3A). Protein kinase D is a family of serine/threonine kinases that play essential roles in directed cell migration9,17. In response to uniformly applied fMLP (red) stimulation, HL60 cells mediate a robust membrane translocation of GFP-tagged protein kinase D1 (green) (Figure 3B and Movie 2). In an fMLP gradient (red) (Figure 4A), HL60 cells actively recruit PKD1 to the leading edge (Figure 4B and Movie 3). A close comparison of the subcellular localization of GFP in the protrusion of the leading edge indicates that PKD1 localizes at the rear of the leading edge (Figure 4C).
<img alt="Figure 1" src="/files/ftp_upload/54511/54511fig1.jpg" /> 
Figure 1. Cell mobility analysis device allows up to 6 simultaneous chemotaxis assays. (A) Scheme shows the design of a cell mobility analysis device chip for simultaneous monitoring of 6 independent chemotaxis assays. Red shows chemoattractant added to the wells. (B) Introduction of HL60 cells to the wells of cells while the chemoattractant diffuses to establish a steady fMLP gradient. <a href="http://cloudflare.jove.com/files/ftp_upload/54511/54511fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54511/54511fig2.jpg" /> 
Figure 2. Simultaneous monitoring of multiple chemotaxis assays with HL60 cells. (A) Montage shows images of cell mobility analysis device chemotaxis assay to examine the inhibitory effects of PKD-specific inhibitors on chemotaxis at times of 0 and 12 min after gradient application. Chemotactic HL60 cells were pre-treated with PKD inhibitor 1 &#956;M CID755673 for 30 min. HL60 cells with or without the treatment of PKD inhibitor were allowed to chemotax in either RPMI1640 starving medium or 100 nM fMLP gradients for 12 min. (B) Scheme shows the travel path length and morphology of traced HL60 cells. (C) Quantification of chemotaxis as total path length, speed, directionality, and roundness. Mean &#177; SD is shown; n = 10, 12, or 11 for no gradient, fMLP gradient without CID755673 treatment, and fMLP treatment with CID755673 treatment, respectively. <a href="http://cloudflare.jove.com/files/ftp_upload/54511/54511fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54511/54511fig3.jpg" /> 
Figure 3. GPCR-mediated robust membrane translocation of PKD1 in response to uniformly applied fMLP stimuli. (A) Multichannel monitoring of PKD1-GFP (green), chemoattractant (1 &#956;M fMLP mixed with 0.1 &#956;g/ml fluorescent dye Alexa 594, red), and DIC (differential interference contrast) to identify the adhering HL60 cells in a well of a 4well chamber coated with 0.2% gelatin in RPMI 1640 medium. Scale bar = 10 &#956;m. (B) Montage shows that uniformly applied fMLP (red) induces robust membrane translocation of PKD1-GFP (green). <a href="http://cloudflare.jove.com/files/ftp_upload/54511/54511fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54511/54511fig4.jpg" /> 
Figure 4. Leading edge localization of PKD1 in chemotaxing HL60 cells. (A) Channel mode acquisition configuration facilitates the visualization of the fMLP gradient and the spatiotemporal dynamics of PKD1. In A-C, HL60 cells transiently expressed GFP-tagged PKD1; to visualize the fMLP gradient generated from a micropipette (DIC), 100 nM fMLP (Red) was mixed with 0.1 &#956;g/ml fluorescent dye Alexa 594. (B) Enriched localization of PKD1 at the leading edge of the chemotaxing cell. Scale bar = 10 &#956;m. (C) Merged images show that PKD1 localizes at the rear of the leading edge in HL60 cells. Green shows PKD1 cellular localization, and the DIC image shows the protruding area of the leading edge. Scale bar = 5 &#956;m. <a href="http://cloudflare.jove.com/files/ftp_upload/54511/54511fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells at JoVE.com

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.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

imaging-g-protein-coupled-receptor-mediated-chemotaxis-its-signaling

Research

JoVE Journal

Biology

10.1K Views.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. 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.

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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Immunology and Infection

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Bioengineering

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Neutrophil Functional Assays for Screening Different Signaling Pathways

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

Author Spotlight: Balancing Speed, Cost, and Accuracy in Neutrophil Function Assessment with NeutroFun Screen Protocol 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood

Source: Yang, K., et al. <a href="https://app.jove.com/v/55615">An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood.</a> J. Vis. Exp. (124), (2017)
This video demonstrates neutrophil chemotaxis assay using an all-on-chip method. The method allows direct isolation of neutrophils from the whole blood by immunomagnetic negative selection followed by chemotaxis assay on a microfluidic chip.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Microfluidic device preparation.

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

Summary

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Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Time-lapse Imaging of Mouse Macrophage Chemotaxis

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

A Microfluidic Chip-Based Method for Rapid Neutrophil Chemotaxis Analysis Using Whole Blood