I have planned to do cell based gene expression (human) study using disease suspected water sample. For that, I planned to use human cell lines.
Still I don't have plan regarding water sample, how can I expose to cell line ? what is the dilution or concentration (considering cell swell or damage)? I couldn't find any articles regarding this. Please help me
You could define this empirically, by culturing the cells in their culture media plus various concentrations of clean water, and see which is the highest concentration that does not effect their viability negatively. This control you will always need anyway, to show that the gene expression alteration you find is due to the pollution in the water, and not to the water (and the decreased cell media concentration) itself.
Hello Karthika.
There are two factors that you should consider in the experimental design.
- 1) Does the agents in the suspected water cause any cell proliferation, ROS accumulation and cell death?
- 2) Does the resulting hypotonic medium enhance the susceptibility of the possible agents in the suspected water to the cells during cell culture?
Because you are planning a test for possible disease agents to the cell during cell culture, you should eliminate any possible or synergistic effects of osmolarity changes in the process of the addition of the suspected water.
My suggestions are as follows;
1) Experiment
Control groups are addition of i) the same vol of sham centrifuged medium to the culture and ii) the same vol of sham centrifuged and autoclaved and millipored water (0.22um).
Experimental groups are iii~vii) the same vol of 0X, 1X, 2X, 4X and 8X diluted, different levels of centrifuged, washed pellet and resuspended in medium.
These will determine the causes of any suspected harmful effects on the cells without much-worried osmotic changes in the final media.
Centrifugation of the water should be performed at 12,000 rpm for 10 min. Remove all the supernatant, resuspend the pellet in the final medium containing FBS, centrifuge it again, resuspend in the same vol of medium as original water vol, and this suspected agents from water is used for the test.
2) Experimnet
You should have another set of experiments containing autoclaved and millipored water control for any possible synergistic effects on the disease as described in a similar way.
Best wishes.
Interesting! It is a challenge when one does not know what are the levels/conc of factors responsible to cause disease.
For effect on human Cells in vitro, I would use 2X culture media (available from Millipore) and dilute the growth media 1:1 and so on with the water sample, or make in-house DMEM or RPMI etc and use the water sample to make the media. The links below should be useful
http://www.bioind.com/support/tech-tips-posters/instructions-for-preparation-of-powdered-medium/
http://www.emdmillipore.com/US/en/product/Dulbeccos-Modified-Eagles-Media-2X-With-4500-mg/L-Glucose-and-L-Glutamine-without-NaHCO3-or-Sodium-Pyruvate,MM_NF-SLM-202-B
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