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MTT assay positive and negative control


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6 replies to this topic

#1 khan N

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Posted 24 February 2019 - 06:54 PM

kindly help

I am confused about the positive and negative control in MTT assay.  when we add DMSO to the positive and negative control well.

 



#2 mdfenko

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Posted 25 February 2019 - 02:19 PM

the positive and negative control assays are identical except for the presence or absence of a component necessary for the reaction to proceed. if that component is prepared with dmso, then the negative control must contain the same amount of dmso. also, since the assay is colorimetric, the dmso may affect color development or optical density and must be accounted for.


Edited by mdfenko, 25 February 2019 - 02:19 PM.

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#3 khan N

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Posted 25 February 2019 - 04:44 PM

thank you mdfnko. but I want to know that when I `Aspirate medium containing MTT` (in 2nd last step) then I will add DMSO to all wells including positive and negative control. OR in the negative control, I will add DMSO when I add medium to the well.



#4 mdfenko

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Posted 28 February 2019 - 03:31 PM

is there dmso in the medium and you're not putting medium into the negative control? then yes, put dmso into the negative control

 

if no, then into all of the wells.

 

if you are still confused then please post your procedure


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#5 khan N

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Posted 28 February 2019 - 04:54 PM

thank you so much for your reply. this is my whole protocol in 96 well plate i will use only 5 well lines means only 24 wells be used.  three lines have 10,20,30, μg/ml drug while the other two well lines will be for positive and negative control.

 

1.    Seeded cells in a 96-well plate (10^4cells/well) in 100 ml medium per well and incubated for 24 h.

2.    Different concentrations (10, 20, 30 μg/ml) of the plant extracts will be added to the cells and incubated for 24, 48 and 72 h.

3.    After 72 h of incubation at 37 ◦C, Add 20 μL of MTT 0.5 mg/mL solution in water into each well. Incubate it further 2 h.

4.    Aspirate medium containing MTT carefully.

5.    Add 0.5 μL DMSO to dissolve formazan crystal into each well.

6.    Absorbance will be measured using a spectrophotometer.



#6 khan N

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Posted 28 February 2019 - 05:01 PM

you mean to say when i Seeded cells in a 96-well plate in 100 ml medium per well then during that time in only negative control media i also put DMSO??



#7 mdfenko

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Posted 01 March 2019 - 02:08 PM

thank you so much for your reply. this is my whole protocol in 96 well plate i will use only 5 well lines means only 24 wells be used.  three lines have 10,20,30, μg/ml drug while the other two well lines will be for positive and negative control.

 

1.    Seeded cells in a 96-well plate (10^4cells/well) in 100 ml medium per well and incubated for 24 h.

2.    Different concentrations (10, 20, 30 μg/ml) of the plant extracts will be added to the cells and incubated for 24, 48 and 72 h.

3.    After 72 h of incubation at 37 ◦C, Add 20 μL of MTT 0.5 mg/mL solution in water into each well. Incubate it further 2 h.

4.    Aspirate medium containing MTT carefully.

5.    Add 0.5 μL DMSO to dissolve formazan crystal into each well.

6.    Absorbance will be measured using a spectrophotometer.

formazan is the product of the reaction. it is insoluble in aqueous media. the dmso dissolves any formazan crystals which have formed. so, yes, you add dmso to all the wells used, including the negative control.


talent does what it can
genius does what it must
i used to do what i got paid to do





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