Comparative analysis of apoptosis measured by Hoechst and flow cytometry in non-Hodgkin's lymphomas

Abstract

Fine‐needle samples of 75 non‐Hodgkin's lymphomas were investigated for apoptosis immediately and after 24 h of culture after in vitro irradiation (2 Gy, 10 Gy, and nonirradiated controls). Apoptotic cells were simultaneously quantified by fluorescence microscopic enumeration of apoptotic cells using Hoechst 33342 staining, and by flow cytometric detection of sub‐G1 peak cells. The nonirradiated controls showed a similar mean percent apoptotic cells using both methods, analyzed immediately (9% by morphology vs. 10% by flow) or after 24 h of culture (40% by morphology vs. 41% by flow). In the irradiated samples, the mean percent apoptotic cells quantified by morphology was higher than by flow cytometry (64% by morphology vs. 55% by flow after 2 Gy irradiation, and 71% vs. 58% after 10 Gy). The results of the two methods were correlated, although large differences were seen between the techniques in individual tumors. In our system, flow cytometric sub‐G1 peak analysis appears to underestimate apoptosis. Of these two methods, we find the Hoechst morphology method to be more reliable for quantitation of apoptosis utilizing fresh fine‐needle sample material, in that discrimination of apoptotic cells from debris is easier and that both early and late apoptotic cells are detectable. Cytometry 32:44–50, 1998. © 1998 Wiley‐Liss, Inc.

REIC/Dkk-3 overexpression downregulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer

“…Hoechst 33342 is an intercalating dye that allows determination of total chromatin quantity variations and the degree of chromatin condensation. 20,21 Using fluorescence microscopy, apoptotic cells were identified by the presence of highly condensed or fragmented nuclei. Apoptotic cells were counted at three to five different fields in microscopic observations.…”

Sinalização celular para apoptose em linhagem celular de adenocarcinoma (MCF-7) e carcinoma ductal invasivo de mama (ZR 7531) tratados com alcalódes isolados de Pterogyne nitens

“…We assumed the hypothesis that caspase 3/7 activity can be evaluated at lower concentrations as function of cell damages, which are not yet totally understood [32][33] Guanidine alkaloids form a rare group of natural products. Although most chemical studies on plant alkaloids show they have phenolic groups in common [34][35] the trisubstituted alkaloid, which was demonstrated to be highly deleterious to the cell lines [35][36]. Comparing the cytotoxicity of the two alkaloids, we observed greater activity in PGD than in PGN, due to the lower IC 50 of the former.…”

“…Entretanto, a avaliação de apoptose por esse método não permite determinar os diferentes estágios de apoptose, denominados precoce e tardia (PEC et al, 2003;BOERSMA et al, 2005). As células em necrose e em apoptose tardia exibem padrões característicos, especialmente quanto à permeabilidade de membrana para o iodeto de propídio que ao ser permebilizado no citosol não podem ser distiguidos (MACIOROWSKI et al, 1998;ELSTEIN & ZUCKER, 1994, PEC et al, 2003. Outra desvantagem do ensaio por citometria de fluxo é o fato de procedimentos técnicos que antecedem a avaliação de apoptose (seleção celular ou centrifugação) enfraquecem a exposição de fosfatidilserina ou aumentam a ruptura de membrana o que poderia levar em resultados falso-positivos (KOROSTOFF et.al., 1998).…”

“…Assim, a seleção e identificação de células em apoptose tardia são mais difíceis de caracterizar no ensaio de anexina V quando comparada com o método de Hoechst/Iodeto (MACIOROWSKI et al, 1998;ELSTEIN & ZUCKER, 1994).…”

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro

“…This process is characterized by various morphological and biochemical alterations that are of crucial importance for embryo development, for maturation of the immune system, and as a defense mechanism against external insults (21) . During apoptosis, alterations occur in the cytoskeleton that induce the contraction and loss of cell volume, chromatin condensation, nuclear fragmentation, and the formation of cytoplasmic vesicles that give rise to structures termed apoptotic bodies (22) . Necrosis is defined as a violent form of cell death initiated by environmental stimuli that results in rapid deregulation of homeostasis.…”