Hey, I am doing electroporation in Hela cells, but somehow most of my cells die, and my transfection efficiencies are not good. It is the first time i do it thus i wonder if it may be "because of my hands". the parameters I use are 1000 V, 20 ms and 2 pulses. i electroporate 50K cells each time with 500 ng of DNA.
With what paremeters could I "play" in order to improve the method?
You have to first troubleshoot the cell death. This is more related to "your hands" whereas the transfection efficiency depends primarily on the electroporation (E) program.
Cells are very fragile after E. So, you want to minimize the stress or manipulations after E. Try the following:
1. Pre-incubate the dish containing fresh culture medium. Transfer the cells to the dish after E and put back into the incubator ASAP.
2. Increase the FBS% up to 20% in the culture medium to make the E cells happy.
3. Avoid air bubbles and do not store the cells in E solution for too long. Essentially do sample by sample from (mixing with E solution, E, transfer to dish).
4. Use special transfer pipette provided by the company and press once to suck all E cells and press another time to transfer the cells to the dish. Do not create bubbles.
5. When you harvest the cells to prepare for E, try using cell scrapper. If you use trypsin, do not over trypsinize your cells (
You have to first troubleshoot the cell death. This is more related to "your hands" whereas the transfection efficiency depends primarily on the electroporation (E) program.
Cells are very fragile after E. So, you want to minimize the stress or manipulations after E. Try the following:
1. Pre-incubate the dish containing fresh culture medium. Transfer the cells to the dish after E and put back into the incubator ASAP.
2. Increase the FBS% up to 20% in the culture medium to make the E cells happy.
3. Avoid air bubbles and do not store the cells in E solution for too long. Essentially do sample by sample from (mixing with E solution, E, transfer to dish).
4. Use special transfer pipette provided by the company and press once to suck all E cells and press another time to transfer the cells to the dish. Do not create bubbles.
5. When you harvest the cells to prepare for E, try using cell scrapper. If you use trypsin, do not over trypsinize your cells (
I assume you are using the Neon, in that case there is an optimization protocol loaded on the machine. In case you want to play with parameters I would advise changing one at a time. It may be worth changing the amount of cells you use. Increasing cell number increases resistance, also try a range of DNA concentration to determine the best amount with limited toxicity. I think for HeLa you could try 1650V, 10mS, 3 pulses or 2000V, 20mS, 1 pulse. Try 1x105, 5x105 and 1x106 cells per transfection and DNA at 500ng, 1ug, 5ug and 10ug. Aim at a cell viability of 70-80% and 40-50% transfection efficiency. Some manufacturer say you can get 98% viability and 80% efficiency, I never got such results and in my opinion are quite unlikely. I think the protocol for HeLa is 1005V, 35mS and 2 pulses with 5x105 cells/transfection. How are you assessing viabilty and efficiency?
Also make sure not to use antibiotics in media at least for 48 hours post transfection.
before giving you answers it would be good to know what electrodes or cuvettes you are using and which media are cells in during and after pulse delivery
My lab is using the Neon Transfection System from Thermo Fisher Scientific. For transfected Hela cell line, we use condition like this: 950V, 35ms, 2 pulse. The concentration of cell is 2x10^5. After transfection, you must wait at least 8 hrs for cells to attach to the bottom. If you are using the same company kit, u can use this condition, it works well for transfect 4 different plasmids.
We are electroporating suspension cells which are very hard to transfect with Biorad machine GenePulser II. I think that the Voltage you are using is too high (but you have always to optimized the condition). In our hands the settings 90-100 V, 975-1000uF, 0.2 cm cuvette, 5.10exp6 /200 ul of electroporation buffer and 10ug of the plasmid are fine. We also before the electroporation wash the cell in PBS and then resuspend in Electroporation Buffer (Sucrose150mM,KH2PO4 5mM,HEPES 25mM, MgCl2 5mM, EGTA/EDTA 2mM,DMSO1%,Glutathione 2mM ) you can also add ATP but it is not neccesary. Two pulses are not necessary (does not increase the effectivity). The puls should be around 60-80msec.
However, I have a question, why you are not using transfection reagents such CaCl2 -is the cheapest, for Hela should be sufficient. If not, you can used Lipofectamine3000. I think this would be the best way to go for transfection since this methods should be more effective in the meaning of cell death, stress/ transfection efficiency.
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