3 replies to this topic

[santorini]

Dear all,

I am planning to construct a puromycin kill curve for my hepatocellular carcinoma cell lines, however, looking at the protocol that I obtained online, I am not very sure of one of the steps, hopefully someone out there can enlighten me! Thanks very much in advance.

After adding different dilutions of antibiotics, we have to monitor the cells everyday until the antibiotics kill 100% of the cells. May I know how can we count the number of surviving adherent cells everyday without detaching them? Is counting cells the conventional way of doing a kill curve? If not, what is the normal way of doing this? Is it roughly estimate the percentage of surviving cells?

Besides, has anyone tried using puromycin on HCC cell lines e.g. SNU-398, SNU-182, HuH-7 etc. before? May I know roughly the optimal effective concentration of puromycin for these cell lines?

Million thanks!

[Dr Teeth]

santorini, on Nov 18 2009, 07:11 AM, said:

Dear all,

I am planning to construct a puromycin kill curve for my hepatocellular carcinoma cell lines, however, looking at the protocol that I obtained online, I am not very sure of one of the steps, hopefully someone out there can enlighten me! Thanks very much in advance.

After adding different dilutions of antibiotics, we have to monitor the cells everyday until the antibiotics kill 100% of the cells. May I know how can we count the number of surviving adherent cells everyday without detaching them? Is counting cells the conventional way of doing a kill curve? If not, what is the normal way of doing this? Is it roughly estimate the percentage of surviving cells?

Besides, has anyone tried using puromycin on HCC cell lines e.g. SNU-398, SNU-182, HuH-7 etc. before? May I know roughly the optimal effective concentration of puromycin for these cell lines?

Million thanks!

You can visually assess the survival rate or if you want more precise data, prepare multiple wells for each concentration of puro and harvest 1 well each day, count the cells, and compare to controls.  I don't know any data for your cell lines of interest, but for many of the lines I have used, ~2 ug/ml puro was sufficient.

[santorini]

Thanks Dr. Teeth! I guess I can just visually assess the % of surviving cells everyday and make a rough estimation. Thanks for giving an idea of the concentration of puro too  

[refolder]

Tryphan blue staining.