Help wanted for T-cells assay

Grace Ho EHO17 at alpha2.curtin.edu.au
Thu Apr 10 23:01:44 EST 1997


Hello everyone,
				My name is Grace and I am currently undertaking fulltime PhD in 
Curtin University in Perth, Western Australia.  My project involved the 
immunological response of mice to systemic candidiasis.
				However, I am having a lot of trouble with my T-cells culturing 
technique.  The source of my T-cells are from the spleens of these 
infected mice.  Once isolated, I had to culture the cells in the 
presence of different proteins isolated from the C. albicans.  Of 
course, I have also included Con A as a postive control and just BSA as 
the negative control.  
			The problems started here.... I am using MTT assay instead of the 
good old Thymidine assay.  The thing is that my MTT are not converted by 
my poor cells.... I have checked their viability and they all looked 
O.K..... but sadly, no MTTs were converted and if they were..... No 
differences were seen between the negative and the positive Con A 
wells...... (I have done this more than 10 times now.. I was previously 
working with Alamar Blue assay... but that was even worse).  
			
Ingredients:
RPMI 1640 (10% FCS, and other essential solutions)
Spleen cells
Various proteins of C. albicans in PBS
MTT (5 mg/ml) 
Cell numbers (4 X105 cells per well)  (Total volume = 200 microlitres)
in 37 degrees celsius at 5% carbon dioxide

			Anyway, I have read somewhere that T- cells do not really 
proliferate, that is why the MTT are not being taken up and 
converted.... but other researchers have no trouble at all.....  Which 
with my misfortune.... doesn't help me a great deal...
			So, can someone help me.... (I am desperate!!!)  :(  Any other 
interesting ideas on T-cell culturing would be great as well!!!!


Thanks....!!!!!  It's much appreciated!!!!



Grace HO
Curtin University of Technology
Perth, Western Australia
Email:  EHO17 at alpha2.curtin.edu.au



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