Our human neuroendocrine cells grow in suspension on uncoated standard tissue culture dishes. They grow in characteristic clumps. Transfection of these cells after dispersion by pipetting has been poor - by nucleofection, transfection reagents or virus. Basically, they do not want to take up virus. Spin-fection etc. doesn't work either. Note these cells form huge (relatively speaking) clumps within 24hrs, a characteristic of many such NET cells when growing actively.
Is it better to grow these cells on poly-L-lysine or other such coated plates? Or is there a preferred method? We have not tried to grow them on such plates yet, but I assume they will attach.
I have no experience of using NET cells, however, I have used promyelocytic HL-60 suspension cells. These cells like other suspension culture also have transfection problem. However, I did Adenovirus transfection using polybrene, which improved viral transfer up to some extent. There are many other such chemicals like protamine sulphate etc which increase viral delivery in the cells. The well known electroporation could be another option....
I transfect SCLC cell lines with Lipofectamine RNAiMAX of Life tech for siRNA transfection. It works very well. SCLC cell lines are thought to be of neuroendocrine origin (there are strong indications about it despite no clear evidence). They grow in suspension, several of them as unshaped aggregates, other as spheres which resemble neurospheres. If you are interested in siRNA approach, I would do a try with RNAiMAX. In order to check transfection efficiency, I use the GLO siRNA of dharmacon.
Best!
Thanks Muhammad and Francesco !
@ Francesco: Any special growth conditions with the cells ? How do you deal with the aggregates for transfection? Do you just disperse the cells and transfect?
2. What transfection efficiency do you see?
(1) I use complete media with FBS, without antibiotics. For what concerns the aggregates, I proceed in this way: (1) gentle move from my flask to a 50mL falcon tube the cells, (2) wait 5-10 minutes to let precipitation of aggregates, (3) remove supernatant, (4) resuspend in some mL of fresh media (1-2mL), pipet with a p1000 many times, like 20 times, to break aggregates. Then I add some other mL of media and I proceed as normal.
Depending by the experiment, I get 80-95% of transfected cells. In the image, you see GLO siRNA in green.
Best,
Ooh, nice !!
Will try........
Can you specify siRNA to cells ratio? uM of siRNA vs cell number?
Also, volume of transfection?
Many thanks.
I do a first step of transfection setting for each cell line in a 24 well dish only with GLO siRNA. These are my variables: (1) 25.000 cells or 50.000 cells. (2) 12.5nM, 25nM and 50nM of siRNA. (3) 0.5, 1.0 or 1.5 microliters of RNAiMAX. Final volume is 600 microliters. Then, once I found my best protocol, I do next steps in 6 well dish, multiplying everything x5.
For the SCLC cell lines that I tested, 50nM of siRNA with RNAiMAX 1:600 or 1:400 are the best condition for both cell concentrations. I prefer 1:600 to save material and to avoid putative toxic effects. However, I strongly suggest to do this initial step.
Best,
Anyone else with other ideas please feel free to reply to my question. It is much appreciated.
- Badges
- Science method