Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

1. Maintenance of Macrophage Cell Lines

1. Grow RAW264.7 and J774A.1 cell lines in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 ºC in the presence of 5% CO₂. To help maintain sterility, add 1% penicillin/streptomycin (pen/strep) to the media (although this is not strictly necessary).
2. Critical step: Ensure that the macrophages are growing in a healthy state for efficient transfection and siRNA-induced gene knockdown. Use cells between passages 3 and 10 after fresh thawing, as these macrophage lines exhibit the best transfection efficiency and maintain robust post-transfection survival at this stage; do not use a higher passage number (>20), as transfection efficiency and gene knockdown both diminish greatly.
3. For optimized siRNA transfection, plate cells at 20% confluency and grow one or two days until the cells reach no more than 80% confluence. Overgrown cells will not transfect efficiently.

2. Programming the 96-well Shuttle System for Transfection

1. Start the 96-well shuttle software on the computer connected to the shuttle. Select new parameter file from the top left file menu.
2. Highlight the wells that will be transfected on the displayed 96-well plate schematic. A green box will indicate which wells are selected.
3. Select the program that corresponds to the macrophage cell line being used (DS-136 for RAW264.7 or DS-130 for J774A.1) and select apply, located below the diagramed 96-well plate. 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.

3. Preparing the Reagents for Transfection

1. Prepare the working Nucleofector™ solution by mixing SF cell line 96-well Nucleofector™ solution with the entire contents of the enclosed supplement tube. Allow the mixed reagents to equilibrate at room temperature prior to use. Store excess complete Nucleofector™ solution at 4 ºC for up to 3 months for future use.
2. Pre-warm the DMEM, RPMI-1640, 0.25% trypsin/EDTA, and phosphate-buffered saline (PBS) at 37 ºC prior to preparing the macrophages for transfection.
3. Prepare the siRNA for transfection by thawing on ice. In order to minimize the number of freeze thaws of the siRNA, freeze new siRNAs in aliquots the first time they are used.
4. In initial experiments, use siRNA at a relatively high dose of 2 µM (diluted 1:10 from 20 µM stocks). Subsequently titrate siRNAs that induce robust gene knockdown down to lower doses, although typically higher doses are needed for efficient gene knockdown in macrophages compared to other cell types.
   NOTE: siRNAs are available from many sources; fairly robust gene knockdown can be obtained using siGenome SMARTPOOLs, which are pools of four siRNAs targeting each gene. As a negative control, use any of various non-targeting siRNA pools or siRNAs.
5. To monitor transfection efficiency, transfect either the pmaxGFP control plasmid (provided in the Amaxa kits) or fluorescently-labeled siRNAs in parallel to the experimental siRNAs. Expect 30-50% transfection of the GFP plasmid (assayed by microscopy or flow cytometry the day after transfection) and >99% transfection of fluorescently-labeled siRNA (assayed by flow cytometry immediately after transfection and recovery).

4. Preparing the Macrophages for Transfection

1. Detach adherent macrophages from the cell culture dishes by trypsinization. Aspirate the media and wash the cells twice with 10 ml PBS. After removing the PBS, incubate the cells with pre-warmed 0.25% trypsin/EDTA (1 ml/10 cm plate) for 1-2 min until cells begin to detach. Gently tap the plate and move the solution during the incubation to maximize trypsinization.
2. After the incubation, add 9 ml DMEM + FBS media, mix gently up and down, and collect the cells by pipetting into a sterile tube.
3. Count the number of isolated macrophages using a hemocytometer. Expect roughly 10 million macrophages per 10 cm plate. Calculate the total number of macrophages needed; each well on the 96-well shuttle requires 200,000 cells. Remember to add a few extra wells' worth of cells to allow for pipetting losses.
4. Pipette the calculated number of cells into a centrifuge tube. Centrifuge the cells for 10 min at 150 x g at room temperature. Do not centrifuge too quickly as this will diminish macrophage health and transfection efficiency.

5. Nucleofection of Macrophages with siRNA

1. Using a sterile 96-well round bottom plate (which facilitates mixing while minimizing reagent loss), add 2 µl of each siRNA to each well to be transfected.
   Note: Depending on the number of siRNAs, this can be accomplished during the macrophage centrifugation step.
2. Aspirate the media from the centrifuged cells. Gently resuspend the cells in Nucleofector™ solution SF (containing the supplement solution), using 20 µl per 200,000 cells pelleted. Once resuspended, use the cells immediately for optimal transfection.
3. Transfer resuspended cells into a sterile trough; using a multichannel pipette, transfer 20 µl of the resuspended cell mixture into each well of the 96-well plate containing the siRNAs. Gently mix by pipetting up and down.
4. Transfer 20 µl of the cell/siRNA mixture into the 96-well Nucleocuvette™ plate. Critical step: avoid generating bubbles during the transfer step as this can induce errors during Nucleofection.
5. After placing all of the experimental samples in the transfection plate, cover the 96-well plate with the provided lid and very gently tap on a hard surface to remove any bubbles from the samples.
6. Place 96-well plate with lid into the 96-well Nucleofector shuttle and select 'upload and start' on the bottom right of the program screen. Prior to Nucleofection, follow the program prompt to save the results.
   Note: During the Nucleofection process, wells successfully tranfected will be depicted on the 96-well schematic on the screen by a green circle with a plus sign. A red circle with minus signs indicates an error, most likely due to bubbles or mis-pipetting the correct volume of reagents.

6. Recovery and Plating of Macrophages

1. Immediately after Nucleofection, using a multichannel pipette, add 80 µl of pre-warmed (37 ºC) RMP1-1640 media to each well. Add the media to the side of each well, allowing the media to slowly mix with the transfected cells, which are very delicate at this stage. Do not add the media too fast, as this will greatly decrease viability.
2. Place the 96-well plates with transfected samples and RPMI-1640 into a 37 ºC incubator with 5% CO₂ for 2 min; this is critical to allow the cells to recover prior to further manipulation.
3. Remove the 96-well plate from the incubator and carefully transfer the mixture from each well into a 96-well tissue culture plate using a multichannel pipette.
4. Using a multichannel pipette, add 100 µl of pre-warmed (37 ºC) DMEM+FBS medium to each well.
5. Incubate the 96-well tissue culture plate with samples in a 37 ºC incubator with 5% CO₂ for 24-36 hr (optimize the exact timing for individual siRNAs, although this time range generally works well).

7. Stimulation of Macrophages with LPS

1. Following the 24-36 hr post-Nucleofection incubation, prepare LPS (or other PAMPs) in fresh media. Prepare enough fresh medium for all the wells (200 µl media/well of the 96-well plate plus a little extra for pipetting). Dilute LPS to a final concentration of 20 ng/ml in DMEM+FBS.
2. Carefully aspirate media from the 96-well plate and add the fresh media containing the LPS to the cells.
3. Monitor the response to LPS or other PAMPs at various times after stimulation. 6 hr is sufficient to generate a robust inflammatory cytokine response for cytokines such as IL-6 or TNFα.

8. Monitoring the Induced Innate Immune Response, Gene Knockdown, and Viability in Macrophages

1. To monitor LPS-induced cytokine production, pipette the supernatant from the cells into a 96-well storage plate. Cytokine production can then be measured by ELISA.
2. Once the supernatant is removed, monitor cell viability using fluorescein diacetate (when cleaved by cellular esterases in live cells, this compound becomes fluorescent). Identify siRNAs that target genes that control cell viability using this approach. Note: the transfection process itself should have little effect on viability if performed properly.
   1. Add fluorescein diacetate (5 mg/ml stock in acetone) to each well (1:100 final dilution in PBS).
   2. Incubate at room temperature for one to five min and then monitor fluorescence in the cells using a plate reader (460 nm excitation, 515 nm emission).
   3. Optional: To ensure that the fluorescence in the wells is uniform, lyse the cells using buffers such as RIPA; this provides a uniform fluorescent signal in the well, as some plate readers are configured to monitor only a small portion of the well; if the cells are not plated uniformly, the fluorescein diacetate could give an inaccurate reading.
3. As an alternative to monitoring cell viability, monitor siRNA-induced gene knockdown by qPCR using RNA derived from the adherent cells.
   1. After the supernatant is removed from the cells (step 8.1 above), add RLT buffer (which is used for lysis and preservation of RNA) directly to the adherent cells to lyse them.
   2. Isolate RNA using a RNA preparation kit such as the RNeasy mini-kit, and use qPCR with gene-specific primers to monitor gene knockdown relative to control siRNA treatment. Use primers to Βactin or GAPDH for normalization.
4. As another alternative to monitor other aspects of the innate immune response, monitor the phagocytic ability of the macrophages by adding FITC-labeled E. coli particles directly onto the adherent cells using a kit. Following the manufacturer's protocol, which takes only a few hours, monitor phagocytic uptake using a plate reader or microscopy.