The overall goal of this protocol is to identify the optimum electroporation conditions for transecting primary cell lines using mouse embryonic, fibroblasts, or mfs. As an example, MF cells and nucleic acid of choice are loaded into a 96 well electroporation plate attached to the gene Pulser MX cell system, which allows for the testing of multiple electroporation conditions in a single run. For optimization experiments, cells are cultured, post electroporation, and analyzed for transfection efficiency.
Using epi fluorescent microscopy and flow cytometry, the most efficient electroporation conditions can then be selected for future transfect. Hi, I'm Adam McCoy, senior scientist at the Gene Expression Division at BioRad Laboratories. Today we'll show you a procedure for easily identifying the best electroporation conditions for transecting primary cell lines.
We use this procedure in our laboratory to study gene expression in these cells. Today I'll be using three different passage numbers of mes or mouse embryonic fibroblasts. I'm using different passage number cultures for two reasons.
First, I will show how important cell health is With primary cell lines, even just a few extra days in culture can make a difference to transfection efficiency. Second, I want to show that the process of identifying the best conditions is the same, even though the efficiency of transfection drops off with the older cells. Your experiment probably won't involve different ages of cells.
You can follow the same procedure with your own experimental design. If you want to compare gene expression and control and treated cells, you can first find the best conditions and then do your experiment using the conditions you've identified. Or you can do your optimization and experiment together.
Like I will demonstrate today, I'll be using a GFP encoding plasmid to look at transfection efficiency by flow cytometry. But the electroporation procedure would be the same even if you're doing something different, like introducing irna and looking at gene expression by real-time PCR. So let's get started.
The mouse embryonic fibroblasts, or mets used in this protocol are adherent cells. So the first step in working with these cells is to lift them from the bottom of the flask using standard tissue culture protocols involving trypsin.Ization. After the cells have detached from the flask, neutralize the trypsin with media containing serum and collect the cells in 50 milliliter conical tubes.
Once collected centrifuge, the cells after the cells have been pelleted. They should be washed by resus, suspending them in a known volume of PBS. Once fully resuspended count the cells knowing that a concentration of 1 million cells per milliliter will be needed for electroporation experiments.
Calculate the appropriate number of cells based on the cell count and transfer them to a clean tube. Centrifuge wants more to pellet the cells. Then carefully remove the snat.
Re suspend the cells in electroporation buffer to a concentration of 1 million cells per milliliter and add 20 micrograms per milliliter of the plasmid. The plates are divided up into 96 wells with columns labeled one to 12 and rows eight to H each set of four wells, or well set shares a pair of electrode plates so the cells in those wells experience the same pulse. For example, cells one A, one B, one C, and 1D are electrically connected in a single well set and would all receive the same pulse.
The next set of four wells, two, A, B, C, and D would all receive the second pulse when the protocol is initiated, even though the parameters may be the same, the 96 well electroporation plate is also set up so that the top and bottom half of the plate is divided. So wells, one E, one F, one G, and one H in the bottom of the plate all received one pulse. But it is distinct from the pulse received by wells one, A, B, C, and D on the top.
In this experiment, only half of the plate will be used. The first three wells of each well set contain cells of different ages. The cells in the top row row A have been passaged five times.
The cells in row B have been passaged nine times and the cells in row C have been passaged 13 times. Row D contains electroporation buffer only prior to electroporation. It's a good idea to check your protocol to make sure the settings are correct.
This protocol will deliver six exponential decay pulses followed by six square wave pulses. The exponential decay pulses increase from 200 volts to 450 volts and all use only 350 micro ferrets. Because of the low resistance of the gene pulse or electroporation buffer being used.
The square wave pulses vary in both voltage and pulse duration. The instrument's capacitance setting needs to be matched to the conductance of the electroporation mixture. If using a higher resistance buffer for the electroporation like DMEM, the same voltage range could be used, but with a larger capacitance such as 950 micro ferrets for the exponential decay pulses.
If the optimal capacitance of the buffer you are using is not known, you will also need to vary capacitance in the electroporation conditions you are testing. The next step is to load 150 microliters of cell plasmid mixture into each well being used for this experiment. The fourth well of each well set being used is filled with electroporation buffer.
After loading the plate, place it into the MXL plate chamber and close the lid. Then press pulse to ate the cells. The ator delivers the electric pulses sequentially to each well set according to the conditions specified in the chosen protocol.
It takes a few seconds to electro pray all the cells. Once the pulse is complete, remove the plate from the plate chamber mix sent. Transfer the cells from each well to a culture dish containing prewarm culture medium.
These mes are being grown in DMEM supplemented with 10%FBS control samples that have not been electroporated are taken from the remaining cell plasmid mixture and added to culture dishes containing the pre-war culture. Medium of the same composition as that used with the electroporated cells. Finally swirl.
Then tap the plate to distribute the cells before placing in the incubator for 24 hours at 37 degrees Celsius, 5%carbon dioxide before analyzing transfection efficiency and identifying which electroporation conditions are the best for your experiment. Once optimal electroporation conditions have been identified, there is a choice of two electroporation methods for future experiments. The researcher can either continue to ate cells in 96 well plates or can ate cells in cuvettes one sample at a time.
Cuvettes can only ate one sample at a time and require four times the volume required for one well of the 96 well plates, but individual cuvettes are less expensive and easy to use when only a small number of samples are needed. The gene pulse or MX cell system is designed so that one can use the same electroporation settings in the plates and cuvettes this time. Instead of the plate chamber, use the mxl shock pod for cuvettes.
Unplug the plate chamber from the back of the instrument and plug in the shock pod. Since the shock pod only delivers the pulse programmed for well set, A, B, C, D one, a different program may need to be selected. To select a previously saved program, press home.
Then select option three user protocols. If you're already in the correct user folder, press enter to select user protocols and scroll down. To highlight the correct program, press enter to select that program.
The instrument is now ready. Now that the program is selected, set up the cuvettes. The mixture of plasmid and MEFs is prepared in the same manner as previously shown.
Load each 0.4 centimeter vet with the same volume used for an entire well set. In the 96 well plates. Since a well set is four wells and 150 microliters of either buffer or cell mixture was used per well at 600 microliters of the plasmid cell mixture per vete.
Next place the vet into the shock pod chamber, press pulse to deliver the electric shock to the cells. After electroporation proceed as in the plate method mix, the cells then transfer aliquots to cell culture plates filled with prewarm media set of control. Wells that were not electroporated swirl the plate and tap.
Then leave the cells to recover for 24 hours at 37 degrees Celsius and 5%carbon dioxide before analyzing transfection efficiency after transecting cells and allowing them to recover. Analyze the transfection efficiency qualitatively using epi fluorescent microscopy and quantitatively using flow cytometry cells that have been successfully electroporated and are now expressing the GFP gene appear green under epi fluorescent microscopy shown here is a typical successful result. Viewing the cells under phase contrast allows visualization of both transfected and unresected cells.
Shown here are the cells that were exposed to the lowest voltage electroporation Pulse at 200 volts. The cells are largely confluent due to the high cell density. The same field of view under epi fluorescence shows a number of cells are expressing the GFP marker, but these are only a small percentage of the cells visible in the previous image at 250 volts.
The total number of live cells seen under phase contrast decreases slightly under epi fluorescence. We can see that the number of GFP expressing cells has increased at the highest voltage applied 375 volts. There are fewer live cells visible, however, a large percentage of the remaining cells are expressing GFP.
Which condition is optimal depends on the experimental design. In some experiments, the largest number of transfected cells might be optimal. In other experiments, the highest percentage transfection might be best.
We are interested in the percentage of cells that are GFP positive under each condition and how the percentages vary with cell age flow. Cytometry can provide quantitative information about the transfect results under each of the different electroporation conditions. Shown here are the percentage of cells that are GFP positive in the passage.
Five cells. Under each of the 12 electroporation conditions, the maximum transfection percentage was approximately 80%under the highest voltage exponential decay. Pulse condition six and 70 under the strongest square wave pulse tested condition 12 with the cells passed nine times prior to the electroporation.
The overall pattern of transfection percentage is nearly identical, but with a very slight decrease in the transfection percentages shown. Here are the percentages of GFP cells in the passage 13 cells, which show a marked decrease in transfect percentage relative to the younger cells. The highest transfection percentages were approximately half what was achieved with the younger cells demonstrating the importance of using healthy cells as soon after isolation as possible.
I've just shown you how to use the MXL electroporation system to identify the optimal conditions for electro probating maps or other primary cell lines. When doing this procedure, it's important to remember to use healthy cells as soon after isolation as possible and to use electroporation conditions that are matched to the electroporation buffer. So that's it.
Thanks for watching and good luck with your experiments.
