Leukotriene B four is a pro-inflammatory molecule that mediates its actions via the G-protein coupled receptors, BT one and BT two on a variety of inflammatory cells. The LTB four B LT one pathway has been shown to be critical in several inflammatory diseases including asthma, arthritis, and atherosclerosis. G-protein coupled receptors or gpcr mediate the transduction of extracellular signals that give rise to intracellular responses such as chemotaxis, intracellular calcium release and gene regulation.
Extracellular ligand binding induces a series of confirmational changes that lead to the activation of heterotrimeric G proteins. G-protein then modulate levels of second messenger, such as cyclic adenosine monophosphate, an acetol tri phosphate, and DYL glycerol to monitor leukotriene B four mediated cell migration in real time bone marrow derived dendritic cells or B MDCs are imaged while likened is delivered via controlled flow from a micro pipette. Time-lapse imaging is performed to assess migration.
Using these live cell real-time imaging techniques, our laboratory discovered that leuko trend B four receptor BT one is functionally expressed on dendritic cells. We also identified phosphorylation and beta arrest and dependent mechanisms of internalization of BT one. Dr.Jla and assistant professor in our laboratory who has developed many of these techniques will be demonstrating both live cell migration as well as receptor internalization To prepare bone marrow derived dendritic cells or B MDCs for cell migration assays plate 0.5 million cells in RPMI 1640 culture medium containing FPS recombinant G-M-C-S-F and recombinant FLT three on a 35 millimeter glass bottom dish.
Once plated, incubate the cells for 16 hours at 37 degrees Celsius and 5%carbon dioxide after 16 hours. Add three milliliters of PBS then aspirate. Repeat this wash at least two more times following the last aspiration, add two milliliters of PBS to the plate.
The cells are now ready for the experiment and should be kept at 37 degrees Celsius. Until then, the imaging system used for the experiments in this video consists of A-T-E-F-M epi fluorescent system attached to a Nikon eclipse TE 300 inverted microscope, equipped with a heating stage and a cool SNAP hq. Digital black and white CCD camera.
The excitation and emission wavelengths are controlled with filter wheels and by a Lambda 10 dash two filter wheel controller. Hardware control and acquisition images are controlled by metamorph software. Two micro manipulators are attached to the microscope stage to hold the pipettes, which can either be fabricated according to the instructions in the accompanying text or purchased.
Transfer 1.5 milliliters of a 100 nanomolar stock of the pro-inflammatory ligand leukotriene B four or LTB four into a 1.5 milliliter fuge tube. Backfill the micro pipette by placing the micro pipette tip in the solution and allowing the solution to enter this method of loading will prevent air bubbles from forming at the end of the micro pipette. Next load a one cubic centimeter tuberculin syringe attached to a microfill needle with one milliliter of LTB four.
Then use it to slowly fill a micro pipette from the top attach tubing to the LTB four filled micro pipette. Then connect the micro pipette and tube to a 21 gauge one and a half inch needle attached to a one milliliter syringe also filled with ligand. Now carefully fix the ligand filled micro pipette onto one of the micro manipulators on the microscope stage to perform live cell imaging of bone marrow derived dendritic cells.
In response to the application of LTB four ligand, remove the plated immature b MDCs from the incubator and place them on the heated stage of the microscope. Bring the cells into view using a 60 x oil immersion lens. Find an area in which the cells are not overly crowded, such that migration can be properly monitored.
Remove the lid from the fluoro dish. Then using the coarse manual manipulator, carefully lower the ligand loaded pipette and bring it in. Closes proximity to the cells using the hydraulic fine micro manipulator.
Bring the pipette into focus. Apply a small amount of pressure from the syringe to initiate the slow release of LTB four ligand from the tip ligand should spread slowly into the media by diffusion. Next, using metamorph software, set the image acquisition parameters to acquire a brightfield image every 15 seconds for two hours.
Keep monitoring every five minutes for progression of migration towards ligand. The method described in this protocol was used to determine the dendritic cell migration towards leukotriene B four. Here, mouse bone marrow derived dendritic cell migration towards 100.
Nan Animal or LTB four is seen this time lapse imaging video shows live cell migration of B MDCs towards ltb. Four similar methods can be applied to determine the chemotactic response of many cell types to a variety of chemo attractants. This type of imaging in combination with fluorescent protein tag molecules can be used to track movements and interactions of molecules in real time in live cells.
Here we demonstrate the interactions of fluorescent tag leukotriene B four receptor one with beta arrestin in real time. RBL two H three is a rat basophilic leukemia cell line isolated from Wister rats, which can be obtained from A TCC. These cells do not express many chemokine receptors but have the cellular components to mediate chemokine dependent signaling pathways.
To assess the ligand induced internalization of BT one in real time RBL two H three cells are transfected with BT one RFP and beta arrest in GFP. During time-lapse imaging ligand is added to the plate. Changes in localization of these proteins can be assessed by image analysis.
To study the G PCR R internalization in real time transfect B LT one RFP and beta arrest and GFP into RBL two H three cells according to the instructions in the accompanying text. Then transfer 300 microliters of the electroporated cells and regular growth media mixture into 35 millimeter fluoro dishes containing two milliliters of regular growth media. Place the cells at 37 degrees Celsius in a humidified atmosphere of 95%air, 5%carbon dioxide to allow them to adhere to the bottom of the dish.
After one hour, replace the transfection medium with regular growth medium and place the cells back in the incubator for an additional 18 to 24 hours. Following the incubation, wash the cells two or three times by adding two milliliters of warm phenyl red free RPMI containing 10 millimolar hippies directly to the plate and aspirating the media. Then add another 1.8 milliliters.
Next, place the culture dish on the heated microscope stage. Collect images at 600 x magnification using a 60 x oil immersion lens. Then switch to the appropriate fluorescence filter.
Choose a healthy cell that expresses HBL BT one RFP on the cell surface and capture here the image is captured. Using the RFP filter switch to the GFP filter, make sure that beta arrest and GFP is expressed in the cytoplasm and capture the image. Then examine the merged pseudo colored images to ensure that bleed through a fluorescence which may occur as a result of mist.
Targeting of the receptors does not occur between RFP and GFP. Next, input the parameters for time-lapse imaging Here. 16 bit images are acquired with the camera bending set to one by one.
The red and green fluorescence images are sequentially captured using RFP and GFP fluorochrome filters via an R-F-P-G-F-P beam splatter. Direct the software to capture images at 32nd time intervals for one hour. Set the camera exposure times such that similar dynamic ranges are obtained.
For the fluorescence intensity of RFP and GFP. Collect images for one minute, then at t equals zero. Add the 200 microliters of the ligand directly into the plate without disturbing it.
Continue collecting fluorescence images for 60 minutes. After adding the ligand, as the images are acquired, they will be automatically stored as TIFF images with sequential file names. The files can then be viewed as individual or combined images using the software.
Finally, after all cell images have been acquired, acquire images with plain media no cells using the same settings. Save the files for use as background images to analyze the images. Use the image subtract function in metamorph to subtract the background from actual data images obtained above.
This will account for any background or autofluorescence from the medium. Next, under the file menu, open the final generated stack file option and open the individual stacks of images. Next, under region measurements select eclipse region trace region and draw the regions of interest, select surface and cytosolic regions to measure the fluorescence intensity of RFP and GFP corresponding to BLT one and beta arrestin translocation.
Using the log data option, which automatically links to Excel plop the amount of fluorescence intensities as a function of time. This graph will provide the information about the kinetics of translocation of a given molecule. In the image analysis can reveal the localization of G PCR R and cytosolic proteins and nucleus.
Here expression of BT one RFP on the surface of an RBL two H three cell is seen. This image shows beta rest and GFP localization in the cytosol of the same cell and here p nuke CFP is seen in the nucleus. The color combined image shown here indicates the distinct cellular localization of each fluorochrome ligand induced interaction of surface GPCRs with cytosolic protein can also be assessed here.
Ligand induced translocation of the BLT one receptor and beta arrestin. This video shows live imaging of a cell expressing B LT one RFP and beta arrestin GFP. Upon addition of one micromolar LTB four, the quantification of the translocation patterns can be monitored using metamorph software.
A line scan of fluorescence intensities in a representative cell is shown at T equals zero. BT one RFP is seen at the membrane and beta arrest in GFP is seen in the cytosol. Upon addition of the ligand overlap of RFP and GFP lines is observed suggesting colocalization of the receptor and the beta arrestin.
In addition, the kinetics of protein movement can be determined here. The kinetics of receptor internalization and beta arrestin translocation. Upon addition of one micromolar LTB four was examined.
The fluorescence intensities were measured as a function of time at membrane and cytosolic locations of the cell addition of LTB four ligand to B LT one RFP and beta arrestin one GFP Transfected RRB two H three cells leads to translocation of beta arrestin one GFP from cytosol to membrane observable as the formation of a yellow ring within one minute. Upon interaction with beta arrestin one GFP, the BT one RFP forms a receptor beta arrestin one GFP complex and translocates through the cytosol in the form of endosomes as evident from the appearance of yellow puncta within five minutes. Using these methods, ligand dependent receptor activation status and critical motifs or processes involved in the receptor activation can be determined.
The most important thing in the experiment is to keep an eye on the buffer in the plate while it gets operated. As experiment goes beyond one hour after its development. This technique appeared the the researchers in the field of chemokines and the receptors to explore ocat migration and receptor ligand interactions in various mammalian cell types.
After watching this video, you should have good understanding of how to set up real time imaging experiments involving chemokines and their receptors and their interactions with cytosolic proteins such as beta restraints.
