3H Thymidine assay with non-adherent cells / cell harvester - cell harvester a must? alternatives? (Oct/02/2007 )
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 or alternate techniques can you suggest? Or is a cell harvester a must?
Thanks in advance for your help!
In the old day, people harvested the adherent cells by scraping the wells and transfer the suspension cells into special vial... cocktail was added and then read by a machine.
I never done it... but my previous mentor told me.
Now, because of safety reason, most people havested their cells onto the glass membrane using vacuum cell harvester (either maual or automatic). After adding the cocktail to the glass membrane, it is read by TopCount.
What type of reader are available to you?
Hope this may help.
i used T cells in 3H prolifration assay.....
i think the Automated vaccum cell harvester is important....to trap the 3H on
printed filtermat special from PerKinElmer life sciences….then after drying I
melted a scintillator sheet on the filter …then read by 1450 microbeta plus liquid
Indeed …the reader determined the procedure