In Vitro Drug Exposure Cytotoxicity Testing and Cell Confluency - (Jul/14/2010 )

Hi,

I have a quick question regarding in vitro drug exposures. Currently, I'm trying to test the cytotoxicity of a pesticide (an endocrine disruptor) on prostate cancer cell lines. I'm using MTT assay to check for cell proliferation and viability and I'm doing time-dose exposures.

My question is, how confluent should the cell line be before I start exposure. Currently, I'm waiting till they are about 85-95% confluent. So by the time I reach around 24-48 hours into the exposure time, they end up being 100% confluent. Is this going to lower my sensitivity (since growth rate might diminish if cells are fully confluent)? I'm not too sure...if anyone could provide some insight that would be great.

Currently, I'm testing various concentrations at measuring viability at 12h, 24h, 48h, 72h, 96h and 120h.

Thanks

-cells-

if the treatment is toxic then the only main point to remember is that the start should be with the same number of cells in each time/ dose exposure. I hope that i am right about this

-shldbcrzy-

cells on Jul 14 2010, 04:51 PM said:

start with a confluency that the cells only after 120h are confluent; it is difficult to compare dividing and confluent cells; so, I would compare the exposure to to both dividing (= non-confluent) and confluent cells in different assays

-Inmost sun-

Hi, thanks for the replies.

The thing is I'm actually not 100% sure if this compound will cause cytotoxicity but it should reduce their growth rate as it is a anti-androgenic chemical...so I guess it's best to make sure they're not confluent before the MTT assay is done?

-cells-

cells on Jul 15 2010, 06:20 PM said:

for dividing cells, cytotoxicity is better tested in the non-confluent phase

-Inmost sun-

Great, thank you for the answers.

I just have a quick technical question. I was wondering what's the best way to aspirate media from multiwells without disturbing the cells adhered to the surface of the well. Would it be ok if you just invert the multiwell plate to remove the media (instead of using a pipette) and then add fresh media with the drug treatments?

-cells-

wouldnt inverting the plate cause some pretty messy cross-well contamination? You'd have to be quick!

What sort of plates are you using? Although its not my area I know that there are cell adhering tissue culture plates out there which facilitate pipette washing steps etc, im pretty sure thats the norm.

-Mycobacterial Madman-

I will be using these 24 well multiwell plates soon (https://www.vwrcanlab.com/catalog/product/i...lass_id=5035457) It says they are tissue culture treated for anchorage-dependent cells...think they should be ok.

-cells-

Inmost sun on Thu Jul 15 07:58:11 2010 said:

Hi,

I have a quick question regarding in vitro drug buy e cig
exposures. Currently, I'm trying to test the cytotoxicity of a pesticide (an endocrine disruptor) on prostate cancer cell lines. I'm using MTT assay to check for cell proliferation and viability buy e cigarette
and I'm doing time-dose exposures.

My question is, how confluent should the cell line be before I start exposure. Currently, I'm waiting till they are about 85-95% confluent. So by the time I reach around 24-48 hours into the exposure time, they end up being 100% confluent. Is this going to lower my sensitivity (since growth rate might diminish if cells are fully confluent)? I'm not too sure...if anyone could provide some insight that would be great.

Currently, I'm testing various concentrations at buy electronic cigarette
measuring viability at 12h, 24h, 48h, 72h, 96h and 120h.

Thanks