A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry.
BACKGROUND
Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood.


METHODS
In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3).


RESULTS
After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples).


CONCLUSIONS
We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.
Diaz-Romero
Jose
J
Department of Transplantation, Novartis Pharma AG, Basel, Switzerland.
Vogt
Gerard
G
Weckbecker
Gisbert
G
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis*; D017209:Apoptosis / Q000235:genetics*; D015415:Biomarkers / Q000032:analysis; D001773:Blood Cells / Q000166:cytology* / Q000276:immunology; D017252:CD3 Complex / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002232:Carbocyanines; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004247:DNA / Q000032:analysis*; D003902:Detergents; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods*; D008297:Male; D051381:Rats; D011917:Rats, Inbred Lew; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
D000954:Antigens, Surface; D015415:Biomarkers; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D002232:Carbocyanines; D003902:Detergents; D005456:Fluorescent Dyes; C098830:TO-PRO-3; D004247:DNA