2 replies to this topic

[cotchy]

Hi all,

Have a question about how to graph or present an antibiotic kill curve in my thesis as my supervisor wants all the data to be included, and not just the one concentration i chose to selct for transfected clones.

Basically i have two types to graph

One for neomycin (G148) treatment of primary ovarian cells to establish the LC50 for these primary cells before immortalisation by transfection with a plasmid containing this selection marker -established to select at 100 ug/ml


Secondly one for my now immortalised cells against puromycin  which was a selection marker of a further plasmid transfection.(selected using 500 ng/ml)

I didnt do any cytotox tests in either case such as MTT or NR agaisnt the antibiotics, i just did it visually on appearance of dead - lysed, contracted or floating to living - looked same as control (not an exact science i know but less time consuming)

Basically all the highest concs. i tested had 100 % toxicity(cells lysed or disfigured), all lowest concs had 100 % viability (compared to untreated control) and then there was  two or three concentrations which had between 30 - 90 % toxicity so I chose the lowest conc with 100% toxicity as the best one to to select for succesful transfections.

So whats the best way for me to put the data together? should i just graph the visual viability (in percentage of what i saw) versus concentration?

Should i have used trypan blue on samples from each individual concentration and work it out more accuartely that way?

if i have got the procedure totally wrong maybe someone could make a suggestion as i am continuing to perform both the selections prior to transfection and would really be interested in hearing how others do it.

[Dr Teeth]

cotchy, on Sep 30 2009, 08:44 AM, said:

Hi all,

Have a question about how to graph or present an antibiotic kill curve in my thesis as my supervisor wants all the data to be included, and not just the one concentration i chose to selct for transfected clones.

Basically i have two types to graph

One for neomycin (G148) treatment of primary ovarian cells to establish the LC50 for these primary cells before immortalisation by transfection with a plasmid containing this selection marker -established to select at 100 ug/ml


Secondly one for my now immortalised cells against puromycin  which was a selection marker of a further plasmid transfection.(selected using 500 ng/ml)

I didnt do any cytotox tests in either case such as MTT or NR agaisnt the antibiotics, i just did it visually on appearance of dead - lysed, contracted or floating to living - looked same as control (not an exact science i know but less time consuming)

Basically all the highest concs. i tested had 100 % toxicity(cells lysed or disfigured), all lowest concs had 100 % viability (compared to untreated control) and then there was  two or three concentrations which had between 30 - 90 % toxicity so I chose the lowest conc with 100% toxicity as the best one to to select for succesful transfections.

So whats the best way for me to put the data together? should i just graph the visual viability (in percentage of what i saw) versus concentration?

Should i have used trypan blue on samples from each individual concentration and work it out more accuartely that way?

if i have got the procedure totally wrong maybe someone could make a suggestion as i am continuing to perform both the selections prior to transfection and would really be interested in hearing how others do it.

If you didn't do any cell counts or take microscopy photos to document the cell death at different concentrations of antibiotic, than you don't have any real data to graph or to show.  You should have documented your results with one of these techniques.  If you do have the data in terms of visual estimate of survival at different concentrations (e.g. 30% cell death at 50 ug/ml, 100% at 100 ug/ml, etc.) written in your lab notebook, you can plot this as percent surviving against dose, as long as you state that this was based on estimates from a visual survey.

[cotchy]

yes it was all visual appearance,

but i could tell when all the cells were dead and also tell when all were alive

so its just a matter of plotting the data of survial on visual inspection up to 100% toxicity level using the data i have in my note book.

thats great cheers.