Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

The overall goal of this procedure is to reliably transfect human THP one macrophages with high cell viability and no cell activation. This is accomplished by first pre differentiating THP one monocytes for 48 hours. The second step of the procedure is to detach the premature TP one macrophages.

The third step is to transfect the cells with IRNA or plasmid DNA, using the nucleo factor technology of lonza. The final steps are to reseed and further differentiate the cells. Ultimately, successful gene knockdown or overexpression can be shown through quantitative realtime PCR, Western blot back, and so forth.

The main advantage of this technique compared to other transfection approaches such as lipofection, is that we can achieve high transfection efficiency together with high cell viability, and that we do not alter microphage function and behavior. Unfortunately, this technique is not suited for high sput applications as no more intense transfect can be performed at a time For this protocol. Have cultured THP one cells in RPMI 1640 with 10%FCS and with 1%pen strep L glutamine 24 hours before the transfection split the cells and provide them fresh media.

The next day. Pre differentiate the cells by seeding 10 to 15 million cells in a 75 square centimeter tissue flask with the following additions to the supplemented RPMI medium, 1%non-essential amino acids, 1%sodium pyruvate, 10 nanograms per milliliter, PMA and 50 micromolar beta mercaptoethanol after 48 hours, aspirate the media and replace it with six milliliters of Accutane. One warmed to 37 degrees Celsius to detach the cells.

Incubate them for 30 minutes during the incubation. Prepare sufficient transfection medium and cultivation medium for the post nucleo affection care of the cells. After 30 minutes, check that the cells are detached and look round.

If some cells are still attached, gently pump the flask with a micro pipette or use simple agitation to detach them. Do not use a scraper. Transfer the suspension to a 15 milliliter tube and centrifuge them for five minutes at 300 Gs and at room temperature, remove the supernatant by aspiration and resuspend the cell pellet.

In one milliliter of RPMI, medium prewarm to 37 degrees Celsius, count the cells and make micro refuse tube aliquots containing two to two and a half million cells for immediate transfection. A speedy execution of this protocol is essential. To avoid loss of cell fatality, be sure to have all materials prepared in advance First centrifuge, all the transfection aliquots for 10 minutes at 250 GS and at room temperature while they are spinning down, load all nucleo effector vets with half a microgram of plasmid DNA or one microgram of SI RNA Use highly concentrated dilution so they occupy a minimal volume of the vet.

Now aspirate the supernatant from just one of the cell aliquots resus. Suspend the cells to 100 microliters in nucleo factor solution. Do not let the cells remain exposed to the solution for more than 15 minutes.

Transfer the cells to the Q vet and mix them around with gentle tapping. Then transfect the cells using program Y 0 0 1 on a model two B nucleo factor. Using a disposable pipette, move the electroporated cells to a reaction vial and immediately add half a milliliter of pre-warned transfection medium to the cells.

Now keep repeating the process with each aliquot of cells one by one until they are all transfected. After completing the nucleo affection, transfer each transfection of cells to a well of a six well plate with 2.5 milliliters of warm transfection media. Mix each well thoroughly with a micro pipette.

After letting all the loaded plates incubate for four hours, check the status of the cells under a microscope. After four hours, most of the cells should be adhered to the plate if needed. At most.

Another hour of incubation will provide sufficient sahe working one well at a time. Aspirate away the transfection medium from the adhered cells and replace it with an equal volume of warm cultivation medium. Be careful to avoid detaching the cells during this step.

Now, incubate the cells for as long as needed. If necessary, replace the media every 48 hours plan to apply treatments so that they end when the transfected DNA or RNA is at its peak effect. When treating the cells with effectors, use a serum free medium without PMA and without beta mercaptoethanol.

Be careful to not let the cells incubate for more than 24 hours without serum. The protocol was used to transfect, THP one macrophages with IRNA cellular morphology was assessed. According to flow cytometric measurement.

The rate of apoptosis and necrosis in the cells was low in both transfected and control cultures. However, careful counts revealed that both apoptosis and necrosis were slightly higher for the transfected samples. This could be because transfected THP one macrophages were more difficult to detach from plates than UNT transfected cells.

Overall, transfection rates were above 90%as analyzed by flow cytometry. Using fluorescent S-I-R-N-A transfection efficiency was also confirmed by fluorescence microscopy. The functionality of the transfection was shown by genetic knockdown of IL 10 RB expression.

Using short interfering RNA Once mastered, the transfect can be performed in one to one and a half hours if it is performed properly. While attempting this procedure, it is important to remember that the culture medium has considerable influence on the performance. This is outlined in the text protocol.