The overall goal of this procedure is to knock down target gene expression in DCS by SI RNA Transfection. This is accomplished by first programming the AM Maxin Nucleo effector. The second step of the procedure is to mix DCS and siRNA together and to pipette the resulting cell solution into Nucleo QVE modules.
The third step of the procedure is to place the plate into the amaxa shuttle tray to begin the transfection process. The final step of the procedure is to infect the cells with the virus to activate the interferon response. Ultimately, results can be obtained that show gene knockdown through quantitative R-T-P-C-R and Western blotting.
This technique is valuable for characterizing signaling pathways in dendretic cells and may contribute to the development of dendretic cell-based immune therapies. To program the Maxon 96 well shuttle nucleo factor for DC transfection, open a new parameter file. Select the number of wells you'll be using for standard transfection by dragging the cursor over the 96 well plate diagram.
Use a minimum of three wells to pool for each experimental sample. Now input the program code in part one select F, F and i part two, select 1 68 from the pull down menus. Then from the solution box, select monocyte human next under control option select standard, and then click on apply.
To include a no transfection control, select additional wells from the diagram is required. Then from the control option, choose no program control and click on apply again. After mixing up the nucleo affection solution, allow it to warm to room temperature.
Place the number of nucleo vete modules needed into the nucleo VETE plate in the correct orientation as shown here by inserting the first one into rose one and two and then so on for all the following tubes. Transfer enough DCS from a cell culture flask into a 50 milliliter tube to have 500, 000 cells per well for the transfection centrifuge the cells for 10 minutes at 400 G at four degrees Celsius and then carefully remove the supernatant. Next, add nuclear affection solution to the tube and then resuspend the DCS by gently pipetting up and down a few times.
Now label eend DPH tubes according to the specific treatment to be performed, and then divide the resuspended cells into the labeled tubes. Add SI RNA at a final concentration of 0.25 micrograms per 500, 000 cells to the appropriate epi endorf tubes, and then mix the cell suspensions by pipetting. Use non-target SI RNA for the no transfection control sample.
Then pipette 20 microliters of the SI RA DC cell suspension into the nucleo vete modules according to the previously programmed experimental layout, ensuring that the liquid is delivered to the bottom of the well cover the nucleo vete plate with a lid and tap the plate on a hard surface a few times to facilitate the removal of air bubbles to transfect the dcs. Insert the prepared nucleo Yvette plate into the nucleo effector 96 well shuttle tray. Then click the upload and start button.
Follow the progress of the transfection process on the display. A black cross on a green background signifies a successful transfection in that well, whereas a black bar on a red background means it was unsuccessful while the cells are being transfected. Prewarm DC growth medium upon completion of the transfection process, remove the plate and add 80 microliters of the prewarm DC growth medium to each well.
Using a multichannel pipette now incubate the plate for 10 minutes at 37 degrees Celsius and 5%CO2. During the incubation period, add 100 microliters of the prewarm DC growth medium to matrix tubes in the same orientation as the nucleo cuvette plate set up. After the incubation period, transfer all 100 microliters of the cell suspensions from the nucleo CVEs into the prearranged matrix tubes.
Then remove and discard those tubes where transfection did not occur. Finally, incubate the matrix tubes for 24 hours or other desired time interval. Label einor tubes for each of the incubating matrix tubes.
Then transfer the matrix tubes from the incubator to a cell culture hood and pool the matrix tubes for each experimental sample into the pre-labeled eend dfs. Next, pellet the cells gently by spinning the eend DPH tubes in a desktop centrifuge for 10 minutes. At 400 G, remove the snat resuspend the cells in serum free growth medium containing Newcastle disease virus or NDV at an MOI of one loosely.
Cover the epi endorphins in a sterile fashion and then incubate the tubes for 45 minutes. After this incubation period, add 900 microliters of DC growth medium and re incubate the tubes for another eight to 10 hours. To harvest the transfected and infected dcs.
Pellet the cells by spinning the einor tubes in a desktop centrifuge as before and remove the snat monocyte dcs were transfected with either IRNA targeting RIG I or non-specific glow irna and infected with NDV as indicated by a plus sign or remained uninfected as indicated by a minus sign as detected by quantitative R-T-P-C-R-A knockdown of R RGA by 75%At the transcription level was observed a similar reduction in the expression of interferon beta. A downstream effector of RGA in the interferon signaling cascade was also observed. Furthermore, the expression of interferon beta in non-infected control transfected cells was not detectable.
While that of MXA and interferon beta downstream response gene was minimal.Here. Data from a second transfection of DCS with rigi targeting irna performed as in the previous figure is shown in this experiment. However, all the cells are infected with NDV and an additional control Using unresected Monocyte DCS was incorporated.
The gene silencing results were similar to those observed in the previous figure. A western blot probed for RGA revealed that the protein expression of this gene had been completely blocked. Lanes one and two show data for lysates from unresected cells.
Lanes three and four show lysates from glow irna transfected cells and lanes five and six show lysates from cells transfected with RIG I targeting S Irna Once mastered, this technique can be done in one hour, including knocking down of many genes simultaneously.
