Do you have any other ‘maintenance’ antibiotics in your medium in addition to G418 (e.g. ‘Pen-Strep’)? According to Invitrogen Pen-Strep is a competitive inhibitor of G418 and sometimes low or no selection is observed if all three antibiotics are being used simultaneously.

The only way to do it is with a kill curve for your cells. Make sure your cells are subconfluent and perform a dose reponse to see which concentration kills all your cells. There is really no other way around this.

I used to work a lot with G418 (25 years ago); I remember that stock solutions were not kept at 4°C, most likely at -20°C.

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Article A model for primitive neuroectodermal tumors in transgenic n...

Hi, I'm agree with Robert and Neil because first you must perform kill curve to adjust the concentration according to your cell line. For example, for human fibroblast BJ cells I'm using 1mg/mL and always keeping stock aliquoted at -20C. However, I'm not sure whether you did let them grow enough time under Neo selection. Normally, puromycin selection occurs quickly 2-3 days because your arrest translation which is much stronger effect than another kind of selection such as hygromycin B or G418 (neo). Be aware if you are performing selection with plasmid that the cells keep as episomal form, because at the time that you are growing cells your plasmid will dilute over time. Based on my experience, BJ cells under Hygro or Neo selection take at least one week to reach 100% dead cells in control cells (without selection gene), similar like Blasticidin. So, I suggest you perform kill curve with different G418 concentrations as you tested growing until you reach 100% dead cells in untransfected cells. After you should use for your experiments the minimun concentrations of G418 that is able to kill unstransfected cells. Good Luck!

...I just realized that you keep DMEM plus G418 at 4C. When I said that storage G418 at -20C it is stock solution (G418 powder resuspended in water, filtered and aliquoted at 250-500 mg/mL). When I add G418 to DMEM I add it at 1mg/mL final concentration and I keep at 4C max 1 week (usually I prepare fresh DMEM plus G418)....

Do you have any other ‘maintenance’ antibiotics in your medium in addition to G418 (e.g. ‘Pen-Strep’)? According to Invitrogen Pen-Strep is a competitive inhibitor of G418 and sometimes low or no selection is observed if all three antibiotics are being used simultaneously.

As Jorge pointed out, G418 needs a couple of days to kill the nonresistant cells (I remember about 10 days to pick resistant colonies, so there should be some killing after a week). If I remember right, I used 1mg/ml final concentration. So you mentioned you used already several concentrations which should work, but you did not mention for how long..., but maybe your batch of G418 is to old.

One issue, that your antibiotic is old to work, as it mentioned. Next, that CHO is a quickly growing line, you should dilute the transfected cells well to get killing. In overgrowing wells or dishes you can not get effective selection. Do some diluted sample from the transfected dish into a 24- or 6-well plate (in selection medium) to check whether your antibiotic works. But variate cell number, ie by stepwise dilution from a starter. And keep the original transfected cells in culture until you decide or get some killing and selection.

Thank you Neil Bradbury. I am running now the kill curve, but still that G418 don't has a significant effect on the cells. It was more than 7-8 days, and they reach confluence after 3-4 days (of 60-70% original seeding).

Thank you Robert Eibl. Yes, the stock has been kept in 4°C, for about one year. Interestingly, it works well with other cell lines, but not my CHO cells. Six well plate seeded with 70% confluence cells, and after 2 weeks still the control and transfected cells growing continuously and both confluence. Thanks for the paper, it is interesting.

Thank you Jorge Vera-Otarola for your explanation and suggestion. As all colleagues here mentioned, there are different factors could interfere and effect either G418 or the cell line itself. I believe using new G418 could has a different outcome; CHO cells really resistance to Neomycin under various conditions.

Thank you Michael Menze. I don't use any other maintenance antibiotics like Strp/Pen. I was wondering if there any possible inhibitor or G418 efficiency effector could be in the DMEM (inorganic components); or with the FCS. On the other hand, adding the G418 in dropwise fashion directly on the replaced medium rather than using medium with previously prepared with G418 - is this point worth to mention?

Thank you Attila Lehotzky. I'll try the 24 well plate, that's to run along with my transfection. Hopefully to have a good results soon.

We use Geneticin (Gibco, cat. no. 10131-035) at a concentration of 400 ug/ ml in our CHO lines.

Thank you very much all of you. I'm considering all related approaches, and hopefully to get this G418 working well in my experiments.

Hi, I also need to get a stable transfected cell line using G418, I facing same problem with you, after trasnfection, there is no selection on cell, because my untransfected cell  is growing well in 1mg/ml G418. How you manage to solve it?i using HCT116 colon cancer cell.

Hi Sue, the fact behind the resistance of CHO cells to G418 is unknown until now. However, I have used the same cells but from different source, and I got new patch of G418. Now, everything is going all right. 

Hi Ibrahim, I am currently facing the same problem with G418, although the batch I have used was new! I am considering changing my G18 batch. could you please tell me where did you order your new G418 from? many thanks 

Hi Narmeen, the G418 is from Gibco (now within FisherThermo) and the details as follow:

Gibco G418 Sulphate

Potency: 713ug/mg

Ref # 11811 - 064

Good luck!

Hi sue ping Eng,I also use HCT116 colon cancer cell,can you tell me the proper concertration of G418 to select this untransfected cell?Thanks.

It varies according to the cell type that you are selecting. For most cell lines it ranges between 500-800 ug/ml 

It varies with cell type. 400ug/ml will decimate even some Osteosarcoma cells. Killing curve determination first!!!! 

I did a killing curve last year and the minimum killing concentration for CHO cells was 2mg/mL (G418). Complete cell death will be observed after 5-7 days. I selected stable clones by adding G418 2mg/mL 8h after transfection.

A question: how do you guys prepare the stock solution of G418, as a normal dilution, or referring to the potency dilution. I could see that a lot of people do the potency dilution, this mean that for 1g of G418 if I want a 50mg/ml stock normally I have to add 20mL of water. However, if you refer to the potency that from sigma is 0.45, doing the potency calculation, 1/0.45 x 50mg/ml = 111.11mg/ml; 1000mg / 111.11mg/ml = rougly 9.0 ml. This means that you have to add half amount of solvent to have a 100% active stock solution. Which one of these two calculations do you normally do???

I used concentration-depended calculations, but not the potency. 

Did you dissolve it in MilliQ water autoclaved and filtered??? Did you use as cells HEK-293?

Many thanks

We usually use the tissue culture medium (e.g. DMEM) in which we intend to use G418 on cells growing on similar medium. This is commercially available and not required to be autoclaved or filtered. You can use G418 on different cell line as long as these cells are transfected and could be selected for this antibiotic. 

Here is a link that describes how to optimize antibiotic selection:

http://knowledge.lonza.com/faq?id=254&search=stable+cell+lines

Guideline for Generation of Stable Cell Lines-Technical Reference Guide

The pdf is attached below.

https://www.creativebiomart.net › resource › principle-pro..

Thank you all for your comments and suggestions. We have used CHO cells from a different source and it works well!

Just to clarify a few things, all G418 stocks are kept in -20 (not 4C as mentioned in the question).

Also, I would appreciate if Michael A Menze could provide us with references regarding the competitive effect of Pen/Strep against the G418 in the DMEM medium.

Cheers,

Ibrahim