Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

The overall goal of this procedure is to transfect Luciferase reports constructs into raw 2 6 4 0.7 cells without activating the cells. This is accomplished by first harvesting raw 2 6 4 0.7 cells from a stock plate by gentle pipetting and seeding cells into a 24 well tissue culture treated plate. The second step is to cot transfect the Firefly luciferase report of plasmid and the control ran luciferase plasmid into the cells.

These plasmids need to be endotoxin free. Next, the cells are stimulated with the desired treatments followed by cell lysis and transfer to a white 96 well plate with a clear bottom. The final step is to assay firefly luciferase activity and then ran luciferase activity to calculate the firefly to ratio.

Ultimately, the fold change compared to the unstimulated control is used to show the effect of stimuli on the Lucifer reporter, which is correlated to the expression level of the gene of interest. The main advantage of this technique over existing methods like conventional overnight transection, is that the transect cells are maintained at their resting state with minimal damage to the cells. The plaid DNA to be used for transfection was previously extracted using a commercially available maxi prep kit and re suspended in 500 microliters of te buffer.

Since the presence of the bacterial cell wall component lipopolysaccharide or LPS interferes with transfection residual bacterial contaminants must be removed from the DNA in a fume hood. Add 500 microliters of phenyl chloroform isoamyl alcohol to the plasmid DNA and shake vigorously for 15 seconds. Use caution because phenol causes severe skin burns.

Incubate the mixture for five minutes of room temperature centrifuge at 13, 000 times G for 10 minutes of room temperature transfer 350 microliters of the upper aqueous phase into a new 1.5 milliliter collection tube. Add 350 microliters of TE buffer to the tube that contains the lower organic phase. Shake vigorously for 15 seconds.

Centrifuge at 13, 000 times G for five minutes. At room temperature transfer 350 microliters of the upper aqueous phase to the same 1.5 milliliter collection tube. Add 70 microliters of a sodium acetate solution and mix well.

Add 700 microliters of 100%isopropanol. Mix well and incubate for 10 minutes of room temperature centrifuge at 13, 000 times g for 10 minutes of four degrees Celsius. Remove the supinate and add 500 microliters of 75%ethanol to the pellet Vortex the samples to mix and centrifuge at 5, 000 times g for five minutes of four degrees Celsius, remove the supernatant.

The plasmid DNA is in the pellet. Open the cap of the tube and air dry the pellet of room temperature for five to 10 minutes. The pellet should become translucent.

Add 500 microliters of TE buffer to the DNA pellet and heat at 50 to 60 degrees Celsius for 10 minutes to dissolve the DNA pellet. Lastly, measure the absorbance of the DNA suspension at 260 nanometers and 280 nanometers with a spectrometer. To calculate the concentration of the DNA and to assess its quality, the raw 2 6 4 0.7 macrophage cells occulted in DMEM supplemented with 9%fetal calf serum in a 37 degrees Celsius, 5%carbon dioxide incubator and passaged every two days.

During each passage, replace the old media with a smaller volume of fresh media and detach the cells from the plate by gentle continuous pipetting seed. 1.5 to 2 million cells on a 10 centimeter tissue culture treated dish as the stock on the day of transfection seed. 200, 000 cells per well.

In a 24 well plate in a volume of 500 microliters of DMEM 9%FCS incubate the cells for four hours in the 37 degrees Celsius incubator. To begin this procedure, warm up the transfection reagent to room temperature in the dark warm warmup, serum free media and DMEM 9%FCS to 37 degrees Celsius. Add 0.5 micrograms of plasmid DNA to 50 microliters of serum free media in a 1.5 milliliter tube.

Add two microliters of transfection reagent with a pipette tip below the surface of the liquid vortex for six seconds. Leave the reagent DNA mixture in the dark at room temperature for 30 minutes. During the 30 minute incubation, remove the media from the wells of the 24 well plate and add 250 microliters of fresh DMEM 9%FCS to each.

Well return the plate to the incubator after 30 minutes. Add 250 microliters of DMEM 9%FCS to the reagent DNA mixture and mix well by puring Up and down, add 300 microliters of the diluted mixture to each well of the 24 well plate incubate for two to four hours in a 37 degree Celsius 5%carbon dioxide incubator. After two to four hours, remove the transfection solution and add 500 microliters of DMEM 9%FCS incubate for 24 to 48 hours in a 37 degree Celsius 5%carbon dioxide incubator.

The begin this procedure by warming up serum free media and DMEM 9%FCS to 37 degrees Celsius. Add 0.75 microliters of the transfection reagent to 18.75 microliters of the serum free medium vortex briefly to mix and incubated room temperature for five minutes. Next, add 0.5 microliters of a one microgram per milliliter DNA solution to the diluted transfection reagent incubated room temperature for 10 minutes.

During the 10 minute incubation, remove media from each well of the 24 well plate and add 250 microliters of fresh DMEM 9%FCS to each well after 10 minutes. Add 250 microliters of DMEM 9%FCS into the reagent DNA mixture. Add 270 microliters of the diluted mixture to each well of the 24 well plate incubate for two to four hours in a 37 degrees Celsius and 5%carbon dioxide incubator.

After two to four hours, remove the transfection solution and add 500 microliters of DMEM 9%FCS to each. Well incubate for 24 to 48 hours in a 37 degrees Celsius and percent carbon dioxide incubator. 24 to 48 hours after transfection, the cells can be stimulated and assayed for luciferase activity.

Replace the media in each well with 250 releases of fresh DMEM 9%FCS Add 50 micro releases of LPS or LPS plus the anti-inflammatory cytokine IL 10 at the desired concentrations to the wells. Return the plate to the 37 degrees Celsius 5%carbon dioxide incubator, stimulate a two to six hours. Next, remove the stimulation solution by aspiration.

Wash the cells with ice cold PBS and add 200 microliters of one x passive lysis buffer to each. Well rock the plate at room temperature for 30 minutes. After 30 minutes, scrape and transfer the lysate to 1.5 milliliter tubes and remove cell debris by centrifuging at 20, 000 times.

G for 10 minutes of four degrees Celsius. Transfer 40 microliters of the clear lysate to a white clear. Bottom 96 well plate measure the Lucifer signal with a luminometer across the entire visible light spectrum.

According to the manufacturer's protocol, low cytometry analysis of cells transfected with a GFP expressing plasmid revealed that the lipid-based reagent gave a transfection rate of about 25%while the protein polyamide based transfection resulted in a rate of about 5%The difference in transfection efficiency was also observed in luciferase signals in cells transfected with the promoter region of I capa b Zeta. The addition of LPS increased the firefly luciferase signal. Typically, the cells are co transfected with a control plasmid containing the ran luciferase gene and the firefly luciferase signals are normalized to the ran luciferase signal.

Comparing the normalized signals between untreated and treated samples showed that LPS upregulates, the activity of the promoter reporter and IL 10 inhibits the effect of LPS. Comparing the length of rest time between transfection and stimulation revealed that luciferase signals decrease over time that this only affected data interpretation. When the protein poly amine based transfection reagent was used to assess the impact of each transfection method on cell death cells were stained with annexin five and propidium iodide and analyzed by flow cytometry only.

The lipid-based transfection slightly increased the proportion of annexin five positive cells. Immuno blatting showed the lipid-based transfection slightly increased the proportion of cleaved poly a DP ribose polymerase protein. While the protein poly based transfection did not.

Despite the elevated numbers of apoptotic cells, the morphology of the cells by light microscopy did not change. More importantly, the biological response of the lipid-based transfected cells remained identical to those of the unresected cells, both made similar amounts of TNF alpha in response to LPS and were inhibited by the addition of IL 10. No TNF alpha was made when the cells were not stimulated, suggesting that the cells remain naive after transfection.

After washing this video user have a good understanding of how to translate raw 2 6 4 0.7 cells with Lucifer reported plasmas in a minimally invasive manner that does not alter the native response of the cells.