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Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose

Nature Protocols volume 4, pages 13131327 (2009) | Download Citation

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Abstract

Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast to the longer term labeling achieved with heavy water (2H2O). As deuterium is non-radioactive and glucose can be readily administered, this approach is suitable for clinical studies. It has been widely applied to investigate human lymphocyte proliferation, but solid tissue samples may also be analyzed. Rate, duration and route (intravenous or oral) of [6,6-2H2]-glucose administration should be adapted to the target cell of interest. For lymphocytes, cell separation is best achieved by fluorescence activated cell sorting (FACS), although magnetic bead separation is an alternative. DNA is then extracted, hydrolyzed enzymatically and analyzed by gas chromatography mass spectrometry (GC/MS). Appropriate mathematical modeling is critical to interpretation. Typical time requirements are as follows: labeling, 10–24 h; sampling, 3 weeks; DNA extraction/derivatization, 2–3 d; and GC/MS analysis, 2 d.

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Acknowledgements

We acknowledge financial support from the Medical Research Council (UK), the Wellcome Trust, Merck Serono and the Charitable Trustees of St. George's Hospital, London, during the execution of studies included in this report.

Author information

Affiliations

  1. Centre for Infection, Cellular and Molecular Medicine, St. George's, University of London, London, UK.

    • Derek C Macallan
    • , Yan Zhang
    • , Hala Ghattas
    •  & Julien Defoiche
  2. Department of Immunology, Imperial College, London, UK.

    • Becca Asquith
  3. Edward Jenner Institute for Vaccine Research, Compton, Berks, UK.

    • Catherine de Lara
    •  & Peter C L Beverley

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Contributions

All authors contributed to the development of the methodology and to the description of the protocol.

Corresponding author

Correspondence to Derek C Macallan.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1: GC/MS chromatogram of the PFTA derivative of deoxyadenosine.

    Deuterium enrichment is determined from the ratio of the M+2 ion (m/z 437, magenta curve) to the M+0 ion (m/z 437, black curve). The two peaks represent cis- and trans-isomers; the larger peak is used for analysis.

  2. 2.

    Supplementary Fig. 2: Example of cell sorting protocol for CD4 and CD8 CD45-sorted “naïve” and “memory” populations using magnetic beads.

    (1) Add CD8 multisort beads to peripheral blood lymphocytes (20 µl per 106 cells); incubate, 10°C, 20 min. (2) CD8+ cells bind to magnetic beads. (3) Wash cells with PBS/BSA/EDTA and pass through a mini-MACS magnet: Retain the CD8 cells which pass through the column for CD4 processing; CD8+ cells remain within the column/magnet. (4) Remove the column from the magnet and flush out retained (CD8+) cells. Repeat Steps 3–4 with a new column to increase purity. (5) Add Multisort release reagent to CD8+ cells from Step 4; incubate, 10°C, 15 min. (6) The release reagent cleaves the magnetic beads from the cells. (7) Pass CD8+ cells through a column to ensure removal of magnetic beads from the cell suspension. (8) Incubate CD8+ cells with CD45R0 beads, 10°C, 15 min. (9) Pass cells through a column: CD8+CD45R0+ cells are retained in the column; CD8+CD45R0 cells pass through. Remove column from magnet and flush out CD8+CD45R0+ cells. Repeat Step 9 for CD45R0+ cells to increase purity. (10) Re-incubate CD8+CD45R0 cells with CD45R0 beads (10°C, 20 min) to ensure removal of any remaining CD8+CD45R0+ cells and RA/R0 double-positive cells. (11) Pass cells through a column. CD8+CD45R0 cells pass through; any CD8+CD45R0+ cells remain in the column. Repeat Step 11 for CD8+CD45R0 cells to increase purity. In parallel for CD4+ populations, take the CD8 cells from step 3, add CD4 multisort reagent, then follow Steps 1–11 as for CD8+ cells to isolate CD4+CD45R0+ from and CD4+CD45R0 subpopulations. (Adapted from Ghattas et al29)

  3. 3.

    Supplementary Material 1: Estimation of dilution factor for precursor enrichment

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https://doi.org/10.1038/nprot.2009.117

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