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

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Help with 3H Thymidine Assay with lymphocytes / cell harvesterrequired?

Help with 3H Thymidine Assay with lymphocytes / cell harvesterrequired? - Protocols and Methods Forum

Help with 3H Thymidine Assay with lymphocytes / cell harvesterrequired? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-02-2007, 07:40 PM
David Liu
Guest
 
Posts: n/a
Default Help with 3H Thymidine Assay with lymphocytes / cell harvesterrequired?



Hello,

I will be doing a 3H Thymidine uptake assay on primary
T cells for the first time. The protocol that was
given to me was for an adherent cell line, and I'm
finding that many people use a vacuum cell harvester
to wash non-adherent cells in the 3H thymidine assay.
Unfortunately, it is difficult for me to have access
to such an instrument, and I'm wondering if it is
possible to do this assay without a vacuum cell
harvester. Does anyone have any experience with
performing this assay on non-adherent cells without a
harvester? Is it possible to just centrifuge and
pipette out supernatant to wash away unincorporated
thymidine and TCA? What recommendations do you have?
Or is a cell harvester a must?

Thanks,
David



__________________________________________________ __________________________________
Tonight's top picks. What will you watch tonight? Preview the hottest shows on Yahoo! TV.
[Only registered users see links. ]

Reply With Quote
  #2  
Old 10-03-2007, 01:18 AM
WS
Guest
 
Posts: n/a
Default Help with 3H Thymidine Assay with lymphocytes / cell harvester required?

Hi David,

basically, washing them many times (I remember having washed adherent
cells in 24 well plates at least 6 times) might do the job, but it is
very likely that you increase the error in your data as you can't
control the number of cells you suck away during that procedure. Maybe
you also want to use any kinds of spin columns (maybe even a pipet
filter tip will do) that can hold back cells and let nucleotides run
through.

What I'd recommend to try is a method that currently used in my lab in
an assay where the binding of radioiodinated antibodies on cells is
measured, so the incorporated radioactivity needs to be separated from
the floating one. It's pretty simple and easy actually: the cell
suspension with the radioactive label is layered on a certain kind of
oil (see below) and briefly spun (maybe 10 seconds) just to drive the
cells through the layer. The whole thing is frozen in liquid nitrogen,
then the tube is cut in the middle of the oil (layer height: 5-10mm)
and the radioactivity in the pellet is counted.

Here comes the below: as you already imagined, the oil serves to
separate the cells from the aqueous liquid, thus the density needs to
be between that of your medium and that of the cells. We use a mix of
dodecane and bromododecane I don't have the ratio at my hands, but I
could dig it out (means ask my student next week when she's back from
holidy).
Meanwhile also google might help you:
[Only registered users see links. ]
But I think, the reference is this paper by Hampe et al.:
[Only registered users see links. ]

The trick is to use thin walled eppis and strong scissors (we use
chinese style, as for trimming Bonsais. Iron sheet cutting scissor
might work as well).

Best regards,

Wolfgang

On Oct 2, 9:40 pm, David Liu <[Only registered users see links. ]> wrote:

Reply With Quote
Reply

Tags
assay , cell , harvesterrequired , lymphocytes , thymidine


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
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
Human Cytome Project - an idea - Update 19 April 2005 Peter Van Osta Cell Biology and Cell Culture 1 06-01-2009 02:17 PM
A Human Cytome Project - an idea - Update 14 March 2005 Peter Van Osta Cell Biology and Cell Culture 0 03-14-2005 02:27 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM
Tissue Engineering & Stem Cell Research 2005 Andrew Oliver Protocols and Methods Forum 0 12-02-2004 11:38 PM


All times are GMT. The time now is 11:03 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.17584 seconds with 16 queries