hi
I have no experience in this field.
1- you should check optimal zeocin cell toxicity concentration before cell transfection (minimal time 7 days).
2- check transfection efficacy with EGFP control plasmid .
good luck
Hello,
if you suspect the transfection reagent to induce tox, 3 things: (1) first use controls, the reagent alone and a GFP control as suggested by Reza Hadavi , (2) try to reduce the amount of reagent, (3) try other reagents, and most importantly, try reagents with different chemistries.
Lipofectamine 3000 is a very powerful reagent but also known to affect cell physiology and sometimes to have tox effects. why? because cationic lipids/liposome-based reagents could interact with all cell compartments and remain in the cytosol. It's difficult for the cells to eliminate this material.
Then, not ideal for stable transfection...
(The Lipofectamines are also very expensive!)
I would recommend to test polymer-based reagents such as our Viromers (in this case Viromer RED or Viromer PLASMID would be the best candidates).
As regards to my tech support experience, I have never get feedbacks for MC-38 cells, then I cannot say if they are particurlarly difficult to transfect, but probably not, since it's a cancer cell line. With our Viromers, we have good results for other colon/rectum cell lines, e.g. HCT116, HT-29, CMT-93... maybe good to give a try... if you are motivated, just write me, I will ship you free samples. I would be happy to learn from you and to add this cell line in our cell database.
Best,
Sandra
Thank you both, very helpful.
I did a Zeocin antibiotic kill curve and have optimized the concentration for complete cell death within 1-2 weeks.
I am planning to try an electroporation method instead of Lipofectamine, and also trying the transfection using different media (pen/strep free) as I think the Opti-MEM reduced serum media wash which is recommended with the Lipofectamine 3000 seemed too harsh on the cells. Even the control with no Lipofectamine looked the same? Very strange as the cells are normally quite adherent and do not wash off with PBS.
With regards to checking transfection efficacy, unfortunately the plasmid we have used cannot contain a fluorescent tag (https://www.invivogen.com/pcpgfree-ova) such as GFP. I am going to test the efficacy using an OVA ELISA and see what result I get.
Thanks again!
hi
You can use pEGFP-N1 as a parallel control in other well. After 24 hours of transfection you can check the cell transfection rate by fluorescence microscope.its critical control to check the transfection efficacy (minimum transfection efficacy for stable transfectant production > 40% )
good luck
I fully agree with Reza Hadavi , you need to test if transfection itself is ok for these cells and your conditions, with a simple GFP (or something else) plasmid, not your plasmid.
Thank you both. The cells were able to be transfected with a GFP plasmid quite easily, but we are still trying to successfully transfect with our plasmid.
- Badges
- Science method