The overall goal of this procedure is to track protein movement by expressing a single protein bearing two different Fluor fours in two separate cell lines and fusing these cell lines together. This is accomplished by first transecting plasmids bearing the gene of interest fused to either of two different fluor of protein genes into two separate cell types. The second step of the procedure is to mix the cells together and allow each cell type to accumulate protein.
The third step of the procedure is to inhibit protein synthesis, then fuse the cells using polyethylene glycol. The final step of the procedure is to visualize protein localization via fluorescence microscopy. Ultimately, results can be obtained that show alteration of protein localization or trafficking behavior through fluorescence microscopy.
This method can help to answer key questions in the cancer biology field, such as how to discrete protein functions and behaviors change as cells become transformed. Though this method can provide insight into transform cell specific changes. It can also be applied to other studies such as cell differentiation, developmental biology, and cell signaling.
Generally, individuals new to this method will struggle because optimization of transfection conditions as well as fusion conditions will be required for each different cell type. The first method demonstrated in this protocol for the introduction of plasmid DNA into cells is the NU Nuclear Affection method, which uses lonza nuclear effector solution to begin wash cells that have been recently passaged and are in logarithmic growth with phosphate buffered saline PBS and harvest them by ization. Once the cells have lost adherence, neutralize the trypsin with the addition of serum containing media and collect cells by centrifugation at 1500 G for five minutes while the cells are being centrifuged.
Add sterilized cover slips to a six. Well play in the tissue culture hood, then pipette 1.5 milliliters of complete culture media into each. Well cover and move the plate to the cell culture incubator to equilibrate.
After centrifugation, move the tubes to the hood, aspirate the super named and resuspend the cell pellets in a minimal volume of PBS. Next, count the cells and aliquot the volume of cell suspension to sterile micro centrifuge tubes. That would provide approximately five times 10 to the fifth cells per sample centrifuge again at 1500 G for five minutes.
Once the centrifugation is complete, place the tubes in the cell culture hood, aspirate the SNA and carefully resus. Suspend the cells in each tube with 100 microliters of room temperature nuclear effector solution. Then add five micrograms of plasma DNA to each sample and transfer the sample to a nuclear effector Q vete and cover.
Take care to avoid air bubbles. Insert the sample qve into the Q vet holder. Select the appropriate nuclear effector program and initiate it.
Selecting the most appropriate program may require prior testing to establish optimal transfection efficiency with minimal mortality. Then plate the cells into the six Well plate in either mixed populations or separately as controls. Return the plate to the incubator and allow the cells to recover for 12 hours.
The second method demonstrated for transient expression of plasmids is transfection with kagen affecting reagent. To begin, add 0.8 micrograms of each plasmid DNA sample to 200 microliters of EC buffer in a sterile fuge tube. Then add 6.4 microliters of enhancer reagent to each sample.
Mix briefly by flicking the tube and incubate for two minutes at room temperature. Following this brief incubation, add 20 microliters of affecting reagent to each sample. Mix each tube by flicking and incubate for 15 minutes at room temperature.
During the incubation, prepare the two cell lines to be used in the experiment. For transfection the cell should be recently passaged and in logarithmic growth at approximately 80%confluence. Wash the cells once with PBS and add back 1.5 milliliters of fresh DMEM with 10%FBS.
Now that the DNA transfection reagents have incubated for 15 minutes, add 1.2 milliliters of DMEM plus 10%FBS media to each sample mixed by pipetting and immediately transfer dropwise approximately 700 microliters of the complexes to each of two wells. In the six well plate mixed by swirling the plate. Allow the cells to transfect for at least three hours in the incubator in the cell culture hood, place one sterilized cover slip per well in a six well plate if desired.
Coated cover slips may be used to enhance cell adhesion. Next, wash the transfected cells twice in PBS and harvest the cells by minimal trypsin ization. This is accomplished by adding 100 microliters of trypsin solution to each well agitating the plate to ensure complete coverage.
Then immediately aspirating the residual trypsin. Neutralize the remaining trypsin by resus suspending the cells in 1.5 milliliters of fresh DMEM with 10%FBS plate the cells in either mixed populations or separately as controls. In the six well plate containing cover slips allow the cells to recover for 12 hours in the cell culture incubator.
Regardless of the transfection method, the procedure for generation of hetero carry infusions is the same. Following transfection, remove the culture media from the wells and wash the cells twice with PBS. Next, it is recommended to treat the cells with cyclo heide to inhibit further protein synthesis.
Add cyclo heide at a concentration of 100 micrograms per milliliter in culture media to cover the cells and incubate for two hours in the cell culture incubator. After two hours, remove the cells from the incubator and wash the cells twice with PBS. To initiate fusion, cover the cells with a solution of 50%polyethylene glycol 1000 in serum free DMEM and incubate for 125 seconds.
At room temperature, remove the PEG solution and wash the cells with PBS to remove residual peg. Then add two milliliters of DMEM plus 10%FBS warmed to 37 degrees Celsius. Incubate the cells for 18 to 24 hours in the cell culture incubator.
Following the 18 to 24 hour incubation. Retrieve the plate from the incubator and wash the cells twice in PBS. Next pipette one milliliter of 4%paraform aldehyde in PBS to each well and gently agitate for 15 minutes.
During the incubation, prepare the microscope slides to be used by adding a single drop of mounting medium containing dpi. Then wash the fixed cells twice with PBS and carefully remove each cover slip with small forceps. Wicking away residual PBS on a piece of tissue before placing it cell side down on a microscope slide with mounting medium plus tappi.
Remove the excess mounting medium from the slide with tissue paper and seal the edges of the cover slips. With nail polish, the cells can now be visualized by confocal or epi fluorescence microscopy. In this flow chart, the hetero method for analysis of cell type specific localization of proteins is shown in three basic steps.
Transfection of the plasmid expressing a fluorescently tagged protein cell fusion with polyethylene glycol 1000 and fluorescent microscopy imaging as can be seen in these epi fluorescence images, primary foreskin fibroblasts, self fused controls expressing GFP tagged chicken anemia virus VP three protein show primarily cytoplasmic localization of the protein in the non-small cell lung carcinoma line H 1299. Self fused controls with the same VP three protein tagged with DS.RED show, primarily nuclear localization. Hetero care infusion of the primary foreskin fibroblast and H 1299 cells results in the introduction of transformed cell material to the primary foreskin fibroblast, and causes a change in localization of GFP tagged primary cell derived VP three to the nucleus.
While attempting this procedure, it's important to remember that success will be highly dependent on transfection efficiencies in the particular cell types being used, as well as optimizing fusion conditions for these particular cells. Following this procedure, other methods like flip and frap can be performed in order to answer additional questions involving real-time analysis of localization dynamics. After Watching this video, you should have a good understanding of how to assess cell type specific protein localizations by introducing fluorescently tagged proteins and generating cell fusions.
