Isolation of peripheral blood CD4(+) T cells using RosetteSep and MACS for studies of DNA turnover by deuterium labeling.
Tracking deuterium ((2)H) incorporation into cellular DNA, after administration of (2)H(2)O or (2)H(2)-glucose, is a recently developed, broadly applicable method for measuring in vivo cell proliferation and turnover that can be used safely in humans. This approach has been used to evaluate the turnover of T-cell subpopulations purified from the peripheral blood of HIV-1-infected patients using fluorescence-activated cell sorting (FACS). A requirement for widespread adoption of this approach for medical decision-making and for use in larger clinical trials is a simple, reproducible, high-throughput method for isolation of highly purified CD4(+) T cells from peripheral blood. Here, we present a simple method, which does not require FACS, for isolating these cells in sufficient purity and yield for analysis of (2)H incorporation into DNA. When blood from HIV-1-infected patients was used, neither the depletion of unwanted cell lineages by erythrocyte crosslinking (RosetteSep) nor the enrichment of CD4(+) cells by immunomagnetic beads (MACS) individually resulted in sufficient purity. The successive application of the two techniques, however, permitted isolation of >95% pure CD4(+) T cells in adequate yield (>10(6) cells/10 ml blood) from healthy donors and HIV-1-infected patients with CD4 counts between 300 and 700 cells/microl. Moreover, (2)H incorporation into cellular DNA after administration of (2)H(2)O to HIV-1-infected patients was indistinguishable between CD4(+) T cells isolated by RosetteSep/MACS and FACS. Thus, both FACS and the new method isolate a similar mixture of long- and short-lived CD4(+) T cells. In practice, the RosetteSep/MACS method is simple, rapid, robust and capable of high throughput.
Busch
Robert
R
KineMed, Inc., 5980 Horton St., Emeryville, CA 94608, USA. rbusch@kinemed.net
Cesar
Denise
D
Higuera-Alhino
Dana
D
Gee
Tracy
T
Hellerstein
Marc K
MK
McCune
Joseph M
JM
D000284:Administration, Oral; D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D004247:DNA / Q000097:blood*; D003903:Deuterium; D005260:Female; D005434:Flow Cytometry; D015658:HIV Infections / Q000097:blood / Q000235:genetics; D015497:HIV-1 / Q000254:growth & development; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods* / Q000592:standards; D007553:Isotope Labeling / Q000379:methods; D008297:Male; D008875:Middle Aged; D012397:Rosette Formation / Q000379:methods* / Q000592:standards
D004247:DNA; D003903:Deuterium