I was used different concentrations (100, 50, 25, 12.5, 6.25, 3.125 µg/ml) of particulate matter (PM10) treated with A549 cell line for 24 hours, 48 hours. I was completed 24 hours reading at 570 NM that values are irrelevant. here with i attach me results. Please help me what are the possibility of mistake I did it.
Hi everybody,
What I meant, sorry I probably did not explain it properly. When you are doing this kind of assay you are looking to a coloration It means you are quantifying an absorbance (optical density) at whatever wavelength you are using. The device you are using has a range between :
- A lower concentration that is not detectable by the device.
- An higher concentration above which one the device is not capable to see any deferences that the saturation point when your reaction is to high for the device to see the deferences.
I did a lot of MTT XTT assay and according to the results provided come with at least two possibilities
- One the compound has no effect at the used concentration and you have no difference with the control.
- The time of incubation with the MTT is too long or the cell seeding is to high (but 5000 cells/wells is usually ok) and even if you have an effect between the compound you reach the saturation of your assay within the 4h of incubation and you cannot see any tiny difference.
As long as there is no positive control as many of you mentioned you cannot determine if the compound as an effect or not.
In my opinion and as mentioned by many of you Senthil has to add a positive control first andr try for higher concentrations of the compound.
Sorry If i didn't explain myself very well in my first message but you have to keep in mind the limits of detection of all the devices you are using that was my point but for sure with all the appropriate positive and negative controls.
Sincerely
Hello senthil where is your positive control because with out positive control u can not say anything about the result .. The given concentration may not be cytotoxic for the cells therefore all the well showing same purple color even though having different concentration . You can try higher concentration of PM10 (higher than 100ug/ml).
Good luck
Hello,
When I looked to your data It seems that your values are to high you are at the saturation level that means that all the values are the sames. When you looked to your pictures you can see that wells are really dark purple that seems to confirm that you are at saturation.
I have few questions:
What are you trying to see proliferation, cell death...?
Does your PM10 is supposed to kill your cells or induce proliferation?
What is your cell seeding concentration?
What are your expected results?
Good luck
Your result is negative that means your compound do not have any cytotoxicity. As deepa said keep one positive control and repeat the experiment again.
Good luck
Thank you guys ... i also accept Deepa taughts,.
Vincent Sarrazy,
i have to see cell death.
PM10 must be kill A549 because it contain PAH compound.
5000 cells/well.
Higher concentration % of viability is low compare to the lower concentration. Thanks
you can use some lower concentration of MTT like 0.5mg/ml depending on the reprints.
you can also study for various time points like 24h, 48h and 72h with reproduciblity.
you can even increase your cell /well as i work with some what more no of cells, again it depends on the reprints elated o your work
regarding your positive control i think you have seeded only cells i rows 7-12, if i am not wrong.
Vincent Sarrazy
i really didnt get you regarding the explanation for your high readings and dark colouration, can you please come up with the explanation again.
Hi everybody,
What I meant, sorry I probably did not explain it properly. When you are doing this kind of assay you are looking to a coloration It means you are quantifying an absorbance (optical density) at whatever wavelength you are using. The device you are using has a range between :
- A lower concentration that is not detectable by the device.
- An higher concentration above which one the device is not capable to see any deferences that the saturation point when your reaction is to high for the device to see the deferences.
I did a lot of MTT XTT assay and according to the results provided come with at least two possibilities
- One the compound has no effect at the used concentration and you have no difference with the control.
- The time of incubation with the MTT is too long or the cell seeding is to high (but 5000 cells/wells is usually ok) and even if you have an effect between the compound you reach the saturation of your assay within the 4h of incubation and you cannot see any tiny difference.
As long as there is no positive control as many of you mentioned you cannot determine if the compound as an effect or not.
In my opinion and as mentioned by many of you Senthil has to add a positive control first andr try for higher concentrations of the compound.
Sorry If i didn't explain myself very well in my first message but you have to keep in mind the limits of detection of all the devices you are using that was my point but for sure with all the appropriate positive and negative controls.
Sincerely
Can i use H2O2 for positive control, Please suggest some of the positive control for PM10 analysis
@ senthil yup you can use H2O2 , DMSO or any detergent like triton-X etc.
Here in our lab we use Triton-X 1% and works fine. You can dilute it in the culture medium. Also, we use a different layout of the plate: line "A": negative control; line "B": positive control; C - H: different concentrations of your sample, from higher to lower. Column 1 and 7 are blanks (only medium).
Look the attachment to see a result. Is this case, you can see that the higher concentrations were apparently close to the positive control, representing citotoxicity. Negative control show a purple color, which means that the cells were alive.
I totally agree Vincent. It looks more like saturation. May be you can first standardize for a positive standard following which you can perform it for ur sample keeping the positive control with it too!
Good luck! :)
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