Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Molecular Biology Techniques
Register Search Today's Posts Mark Forums Read

Molecular Biology Techniques Molecular Biology Forum. Includes forums for common molecular biology techniques.


Question on MTT assay

Question on MTT assay - Molecular Biology Techniques

Question on MTT assay - Molecular Biology Forum. Includes forums for common molecular biology techniques.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 04-06-2010, 03:32 PM
Pipette Filler
Points: 90, Level: 1 Points: 90, Level: 1 Points: 90, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2010
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Question on MTT assay



Hello,
Just wonder if anybody can help me out.

I am using MTT Cell Proliferation Assay for cell growth and cytotoxicity assay. During optimization assay, I am getting quite low absorbance value i.e around 0.3-0.4 for very high cell concentration i.e 10^5 cells/mL.

I noticed:

1. The reaction of MTT with my blank (i.e media only) is quite strong. I am using Leibovitz media supplemented with 10% FBS. The absorbance is around 0.7-0.8. I verified with another media, RPMI 1640 and the absorbance is much higher than Leibovitz. Around 2.0. Due to this I am wondering if I should take out the media before putting the MTT. Do u think this is OK with the assay and will give better absorbance value?

2. After putting MTT, I kept the plates in the incubator for 4 hours. However the formazan formattion was very weak. When I extended to 24 hours it was better, and absorbance reading was higher. Is there any adverse effect eg toxicity to the cells due to the prolonged exposure to MTT?

3. Referring to ATCC determining optimal cell count protocol, the ideal number of cells to be used should yield an absorbance of 0.75-1.25. Do you know why is that figure? Does this apply to all cell lines? I am using colorectal cancer cell lines SW620 and SW480. Will also use few other cell lines i.e SW1116, SW948, CCD-18-Co after this. So far, from my assay, the absorbance values for 3000-7000 cells/well are quite low, around 0.3-0.4.

4. How do you find the optimum wavelength for your assay? I use 550/690nm bcoz it was the recommended wavelength from the brochure. But just wondering whether my low absorbance is due to the wavelength.

Many many thanks ahead for your help.
Reply With Quote
Reply

Tags
assay , mtt , question


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Bradford Protein Assay question klhayes Protein Science 5 01-06-2009 10:58 PM
Bradford Assay Question Denise Protein Science 0 05-15-2008 05:56 PM
Biorad Bradford Assay Deanne Bell Protocols and Methods Forum 0 05-13-2004 03:21 PM
Sci.chem FAQ - Part 1 of 7 Bruce Hamilton Chemistry Forum 0 01-15-2004 09:06 AM


All times are GMT. The time now is 08:26 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12161 seconds with 16 queries