The overall goal of this procedure is to use RNA interference to investigate the effect of candidate genes on the innate immune response. In mouse macrophages to do this, mouse macrophage cell lines are tryps inized and combined with transfection solution and the SI RNAs of interest, the mixture is then transferred to a 96 well nucleo VETE plate and a Maxa 96 Well transfection system is used to transfect siRNA into the macrophages and after an incubation with cell culture, medium, LPS or other activators of innate immunity are applied to the transfected macrophages to stimulate them. Finally, Eliza's are then performed to monitor cytokine production and assess the effect of the siRNA treatment on the innate immune response.
The main advantage of this technique over lipid mediated transfection is that our assay is robust and can be applied to many genes in high throughput profession. The key to this method is to work with healthy cells, treat them gently, and work efficiently during the transfection process. As we will demonstrate, This protocol uses the raw 2 64 0.7 mouse macrophage cell line, which is cultured as described in the accompanying document.
In preparation for the INA transfection plate, the cells at 20%co fluency and grow one or two days until the cells are no more than 80%confluent. It is important to remember to use cells that are between passages three and 10. After thawing, begin this procedure by programming the 96 well shuttle system that will be used for transfection.
To do this, initiate the 96 well shuttle software on the computer connected to the shuttle from the top left file menu. Select new parameter file. Highlight the wells that will be transfected on the displayed 96 well plate schematic.
A green box will indicate the selected wells. Choose the program that corresponds to the macrophage cell line being used for raw 2 64 0.7 cells. Choose DS 1 36 select apply.
Note that after selecting a program, the wells on the schematic plate will be colored. Dark blue empty circles indicate wells that have not been assigned a program and will not be shocked. Next, prepare the working nucleo effector solution by mixing SF cell line 96 well nucleo effector solution with the entire contents of the included supplement tube.
Allow the mixed reagents to equilibrate at room temperature. Warm the D-M-E-M-R-P-M-I 1640 0.25%tripsin EDTA and PBS at 37 degrees Celsius. Prepare the experimental S-I-R-N-A-P max GFP control plasmid or fluorescently labeled siRNAs by thawing them on ice to detach adherent macrophages from the cell culture dishes.
Aspirate the medium and wash the cells twice with 10 milliliters of PBS. Then after removing the PBS, incubate the cells with one milliliter of pre-warned 0.25%tripsin EDTA per plate until the cells begin to detach. This takes one to two minutes.
Gently tap the plate and move the solution during the incubation to maximize trypsin ization. After the cells have detached, add nine milliliters of DMEM plus FBS mix gently by pipetting up and down, and then collect the cells by pipetting into a sterile tube. Count the number of isolated macrophages using a Hema Cytometer.
Expect roughly 10 million macrophages per 10 centimeter plate. Calculate the total number of macrophages needed each well on the 96 well shuttle requires 200, 000 cells. Pipette the calculated number of cells into a centrifuge tube.
Spin the cells for 10 minutes at 150 G at room temperature to a sterile 96 well round bottom plate, which facilitates mixing while minimizing reagent loss. Add two microliters of each IRNA to each well to be transfected. Note that depending on the number of siRNAs, this can be accomplished during the macrophage centrifugation step.
The most difficult aspect of this procedure is the electroporation, the keys that treat the cells very gently and to work very quickly. Once the cells are in a toxic transfection solution, Aspirate the medium from the centrifuge cells, then gently resuspend them in nucleo effector solution. SF containing the supplement solution.
Immediately transfer the cells into a sterile trough using a multi-channel pipette transfer 20 microliters of the resuspended cell mixture into each well of the 96 well plate containing the siRNAs gently mixed by pipetting up and down. Next, transfer 20 microliters of the cell INA mixture into the 96 well nucleo vete plate. It is critical to avoid generating bubbles during this step as this can induce errors during nucle affection.
After placing all of the experimental samples in the transfection plate, cover it with the provided lid and very gently tap it on a hard surface. To remove any bubbles, place the 96 well plate into the 96 well nucleo effector shuttle, and at the bottom right of the program screen, select upload and start. Then follow the program prompt to save the results during the nucleo affection process wells successfully.
Transfected will be depicted on the 96 well schematic on the screen by a green circle with a plus sign. A red circle with a minus sign indicates an error most likely due to bubbles or pipetting the incorrect volume of reagents immediately after nucleo affection. Use a multi-channel pipette to add 80 microliters of prewarm RPMI 1640.
Medium to the side of each. Well allow the medium to slowly mix with the transfected cells, which are very delicate at this stage. Adding the medium too fast will greatly decrease viability.
Place the 96 well plate with transfected samples and RPMI 1640 into a 37 degree Celsius incubator with 5%CO2 for two or more minutes. It is critical to allow the cells to recover prior to further manipulation. After removing the plate from the incubator, use a multi-channel pipette to carefully transfer the mixture from each well into a 96 well tissue culture plate.
Then again, using the multichannel pipette, add 100 microliters of DMEM with FBS to each. Well incubate for 24 to 36 hours immediately after transfection and recovery perform flow cytometry. To assess the transfection efficiency of fluorescently labeled SI irna, more than 99%of the cells should be transfected the day after transfection assay For transfection efficiency of the GFP plasmid, 30 to 50%of the cells should express GFP Following the 24 to 36 hour incubation, prepare LPS or other activators of innate immunity in fresh medium dilute the LPS to a final concentration of 20 nanograms per milliliter in DMEM with FBS.
Then carefully aspirate the medium from the 96 well plate and add the fresh medium containing the LPS to the cells. Place the plate in an incubator to monitor the response to LPS or other stimulators of innate immunity at various times after stimulation. Pipette the supernatant from the cells into a 96 well storage plate for analysis by Eliza.
After removing the supernatant to monitor cell viability, add 100 microliters of a one to 100 fluorescein di acetate in PBS to each well incubate at room temperature for one to five minutes. Monitor the fluorescence in the cells using a plate reader when cleaved by cellular esterases in live cells. This compound becomes fluorescent to illustrate the utility of this approach for monitoring the innate immune response.
siRNAs targeting known innate immune regulatory genes were transfected into the raw 2 64 0.7 macrophage cell line. The cells were then stimulated with LPS and production of the pro-inflammatory cytokines. IL six and TNF alpha was monitored by Eliza as shown here.
Inhibition of toll-like receptor four, the receptor for LPS, but not other TLRs. Using SIR, a strongly diminished production of LPS induced IL six and TNF alpha. Note that inhibition of IL six with irna diminished IL six production, but did not affect TNF alpha production.
These data suggest good efficacy and good specificity of siRNA mediated knockdown of candidate regulators of innate immunity. It is important to remember that the key to a good transfection and good cell recovery is to work with very healthy cells, treat them gently during the transfection and recovery process, and work quickly once the cells are in a nucleo effector solution. Following this procedure, many other assays can be performed including QPCR to monitor gene knockdown and many other assays to investigate the innate immune response.
