Hello Maddie Yeo

Removing the cell culture media and adding the drug concentration in endotoxin-free water is not desirable. This will come as a shock/stress to your cells. The desired drug treatment should ideally be performed in the cell culture medium. If I am not wrong, media like RPMI and DMEM, are they not endotoxin-free? In case they are, you may directly transfer a small amount of your stock solution into your cell culture dishes. Since 10cm dishes are large and would require a lot of media and hence more of your stock solution. I would recommend that you begin with either a 96 well plate, 6/12/24 well dishes, or even 3cm plates (but even this would have at least 1-3 ml of media). Hopefully, this helps you.

Jatin Chadha thank you for your response. So even if the protocol states that the stock solution has to be diluted in endotoxin free water I can just dilute it in cell media if they are also endotoxin free? The drug I am using is Amlexanox from Invivogen.

Hello Maddie Yeo

You need to weigh 10mg Amlexanox in 1ml DMSO. This stock solution can be aliquoted and stored at -20 degree C for at least 6 months.

You can prepare further dilutions either in endotoxin-free water or culture media.

Any reason for using 10cm dish. As mentioned by Jatin Chadha you should use smaller dish like 30mm or 6-well/24-well plate to see the effect of the drug.

For 10cm dish the volume of media is approximately 10ml.

If you use a 6-well plate the volume of media per well is approximately 2ml.

So, to prepare 25uM working solution in 2ml you need to add 1.49ul stock(33.5mM) to 1998.51ul of culture media.

Similarly, for 50uM add 2.98ul of stock (33.5mM) to 1997.02ul of culture media.

for 100uM add 5.96ul of stock (33.5mM) to 1994.04 ul of culture media.

Since you will have to deal with small volumes of stock solutions, there might be errors while pipetting. So, you can dilute your stock solution (1:10) in culture media and then use this diluted stock solution to make your working solution.

So, for 25uM you will have to use 14.9ul of diluted stock,

50uM you will have to use 29.8ul of diluted stock,

100 uM you will have to use 59.6ul of diluted stock.

Make up the volume to 2ml in each case with culture media and gently mix.

Finally, remove the media from the wells and add these working solutions (2ml) containing the drug to the respective wells.

Good Luck.

Malcolm Nobre thank you so much for your detailed explanation and calculations! I think I'll heed your advice and switch to 30mm dishes instead!