The experiment I do is to treat cells with LPS for 24 hours to induce inflammation, then treat them with an anti-inflammatory drug and check for anti-inflammatory cytokines again 24 hours later.
I treat the cells with LPS when the confluency is around 60%, but the problem is that after 48 hours, the cells are completely overgrown and dead.
What confluency do you let the cells reach when you process LPS to induce inflammation?
What is the optimal density?
Is 100% confluency at the end of the experiment the most appropriate seeding density?
Hi Juhee Seo
Which cells are you using for the experiments?
For cytokine analysis, If I'm using RAW 264.7 cells, I would generally count and plate the cells in a 6-well plate (50,000 cells/ well). 12hrs following plating, I expose cells with LPS-1ug for 6-12 hrs and then treat the cells for 24hrs and measure cytokines.
* If your cells are highly proliferative as RAW cells are, so I would recommend to plate at a lower density.
* Since your LPS exposure time is 24hrs, plate at a low density, around 40%. So, at the end if it is around 80-90% that should work well.
Thanks,
Samir Ranjan Panda
Thank you so much for your help!
I have an additional question.
I am growing canine chondrocytes.
I seeded them in 6 wells at a 7.5 x 10^4 cells/cm^2 density.
24 hours after seeding, the confluency seems to be around 50%.
But next 24 hours, the confluency is almost 90%.
By any chance, what is the confluency of your cells when treated with LPS? lower than 50%?
I'd like to try to match my cells to that confluency and treat them with LPS.
I would really appreciate it if you could give me an answer.
The pictures below are 24h and 48h after seeding.
Hi Juhee Seo
I can see the confluency looks good in the images, so you can give treatment 24hrs post seeding the cells and then 24hrs hrs post LPS exposure if the cells are as in the 2nd image, it should work fine.
* Since LPS also enhances the cell proliferation in RAW cells, So I give treatment in around 60-70% confluency state and then post treatment it is around 80-90% confluent.
You can perform the experiment according to the images attached, So that should work.
Thanks,
- Badges
- Science method