2 replies to this topic

[Seng]

Dear all,

FYI, I performed MTT assay for evaluating the anti-proliferative activity of an extract. The protocol is as follows:
1) Plate 100ul cell suspension.
2) Add 100ul extract directly without discarding the spent media (to reduce variation).
3) Add 20ul 5mg/ml MTT solution and incubate for 4h.
4) Add 50ul 10% SDS/0.01M HCl
5) Incubate in 5% CO2 incubator at 37oC for 1h before measuring at 590nm.

Noted: Total solution volume = 270ul. All solution are just added into well of 96-well plate without discarding in order to reduce the OD variation.

Since I never use 10% SDS/0.01M HCl (suggested by my supervisor) as formazan crystals solubilising solution. So, I decided to perform a trial. I plated 100ul cell suspension for 24h. On next day, I added 20ul MTT solution for 4h before adding 27ul 10% SDS/0.01M HCl for 1h in incubator (total solution volume = 147ul). After 1h, I can see the formazan crystals are dissolving in the solution, to enhance the solubility, I placed the 96-well plate on plateform shaker. After 10min, I found that the formazan crystals formed a single clump and did not dissolve at all. Why did this happened? Is it the solution preparation problem? I knew that the normal way is to incubate the formazan crystals in the presence of 10% SDS/0.01M HCl solution for overnight. But, in order to get a faster result, my supervisor suggested me to incubate for 1h and shake a little. I personally think that adding 50ul 10% SDS/0.01M solution might not enough (but the total volume already 270ul in a well), therefore longer incubation period without shaking or shorter incubator period with shaking is needed.

Hoping, someone could solve my doubt.

Regards,
Seng

[EvilTwin]

Hi, I use a totally different protocol. After adding MTT and incubating for a ceratin period (checked by previous optimisation depending on cell type) I aspirate media (dyed cells stay on the plate bottom) and add 100ul of pure isopropanol or isopropanol with HCl (1 ul pf 36% HCl for 10ml of isopropanol). 10 min of shaking is usually enough to dissolve the crystals and in my opinion it's a more reliable method as you measure the signal only from the MTT that was really metabolised by your cells. I have never had any problem.
BTW how do you intend to shake 270ul per well on a 96-well plate without spilling?

Edited by EvilTwin, 17 April 2012 - 02:58 AM.

[tonyqcc]

Hi Seng and EvilTwin.

My protocols are kinda similar to Seng's, to a certain extent.

Seng's protocols:
1) Plate 100ul cell suspension.
2) Add 100ul extract directly without discarding the spent media (to reduce variation).
3) Add 20ul 5mg/ml MTT solution and incubate for 4h.
4) Add 50ul 10% SDS/0.01M HCl
5) Incubate in 5% CO2 incubator at 37oC for 1h before measuring at 590nm.

My protocols:
1) Plate 180ul cell suspension, and incubate for 24h (for cells to adhere to the microplate walls).
2) Add 20ul extract directly, and incubate for 24, 48 or 72h (treatment time).
3) Add 20ul 5mg/ml MTT solution and incubate at 37"C, in dark, for 3h.
4) Remove gently all or most of the media in the wells, and add 200ul DMSO.
5) Agitate the microplate for 15min via orbital shaker or microplate shaker, and measuring ODs at 554nm (with reference at 690nm) within 1h of adding DMSO.

Maybe you can try using DMSO as the solvent? I came across few papers stating that isopropanol may not be a very effective solvent to dissolve formazan salt. No idea for SDS though, cause never heard of it before...

Anyway, hope it helps. Take care~

Tony.