a Hôpital Rothschild AP-HP, 5 Rue Santesse, 75012 Paris, France
b Hôpital Tenon, AP-HP 4 Rue de la Chine, 75020 Paris, France
c Université Pierre et Marie-Curie, 4 Place Jussieu, 75005 Paris, France
Dextran does not penetrate the living cells. However when the plasma membrane becomes permeable, it is concentrated in the cells. This is why we used fluorescein isothiocyanate-conjugated dextran (FITC-Dextran, MW 4000) to identify apoptotic and necrotic cells by flow cytometry. Heat shock was used to induce cell apoptosis or necrosis. To induce apoptosis cells were heated to 43.5°C for 1 hour and then incubated at 37°C. Thereafter the cells were stained with FITC-Dextran or propidium iodide (PI), and analyzed by flow cytometry. FITC-Dextran stained the cytoplasm and/or the nucleus of 80% of the HeLa cells, while annexin V-FITC stained 43% of cells and PI stained the nucleus of 18% of cells. When heated at 50°C and 60°C, the percentages of necrotic cells increased proportionally to heat treatment, 41.7% and 77% of the cells were stained by FITC-Dextran, while 39.5% and 70.3% were stained by PI. FITC-Dextran was selectively internalized across plasma membrane after moderate heat shock. In contrast, in the necrotic cells, the permeability of the membrane was not selective and the percentage of cells stained with FITC-Dextran or PI, was equivalent. Our results indicate that FITC-Dextran can be used as a marker to reveal the cellular damage induced by heat shock and to study the early as well as the late stages of apoptosis
open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
* Address correspondence to this author at the Service d'Hématologie et d'Immunologie Biologique or Service de Dermatologie et d'Allergie, Hôpital Tenon, 4 rue de la Chine, 75970 PARIS Cedex 20, France; Tel: 0033156017343; Fax: 0033156017780; E-mail: email@example.com