Labeling Cells in Microtiter Plates for Determination of [3H]Thymidine Uptake

Ethan M. Shevach1

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3D
DOI:  10.1002/0471142735.ima03ds21
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

A number of protocols in Current Protocols in Immunology use as their end‐point the determination of cell proliferation by determining the incorporation of [3H]thymidine into cellular DNA. This appendix presents a protocol in which the radioactive label is added during the last 4 to 24 hr of the culture. A semiautomated cell harvesting apparatus is then used to lyse the cells with water and precipitate the labeled DNA on glass fiber filters. The filter pads are then dried and counted by standard liquid scintillation counting techniques in a scintillation counter.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

         
     
    GO TO THE FULL PROTOCOL:
    PDF or HTML at Wiley Online Library

    Materials

    Basic Protocol 1:

      Materials
    • 1 mCi/ml [3H]methylthymidine (6.7 Ci/mmol; Du Pont NEN or ICN Biomedicals)
    • Complete RPMI without serum ( appendix 2)
    • Microtiter plates containing cells cultured for 1 to 7 days
    • Repeater micropipet (Brinkmann)
    • 0.50‐ml Combitips, sterile (Brinkmann)
    • Semiautomated cell harvesting apparatus
    NOTE: Use sterile technique throughout and proceed cautiously when handling radioisotopes.
    GO TO THE FULL PROTOCOL:
    PDF or HTML at Wiley Online Library

    Figures

    Videos

    Literature Cited

    GO TO THE FULL PROTOCOL:
    PDF or HTML at Wiley Online Library