Lectinocytochemical detection of apoptotic murine leukemia L1210 cells

Abstract

Background Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about its changes during apoptosis. We investigated whether specific changes in the expression of plasma membrane glycoproteins take place during apoptosis and whether these changes could be used for a quantitative estimation of apoptosis. Methods Lectin cytochemical study of normal and apoptotic murine leukemia cells of the L1210 line was done. Horseradish peroxidase–labeled Laburnum anagyroides bark agglutinin, Phaseolus vulgaris agglutinin, Pisum sativum lectin (PSL), Ricinus communis agglutinin (RCA‐120; 120 kDa), Solanum tuberosum agglutinin, Triticum vulgaris lectin (wheat germ agglutinin), Viscum album agglutinin, Canavalia ensiformis lectin (concanavalin A), and Helix pomatia lectin were used to compare specific glycoprotein expressions in normal and apoptotic murine leukemia cells of the L1210 line sensitive (L1210) and resistant (L1210R) to apoptosis induction by cisplatin. Results The data demonstrated significantly increased binding of α‐D‐mannose–specific PSL lectin (P < 0.01) and β‐D‐galactose–specific RCA‐120 lectin (P < 0.001) by the apoptotic cells of the L1210 and L1210R lines in comparison with the intact cells. That binding was shown to be specific because it was blocked by the corresponding inhibitory sugars. Conclusions Lectins specific to α‐D‐mannose (PSL) and β‐D‐galactose (RCA‐120) can be used to distinguish between native and apoptotic murine leukemia L1210 cells and to quantitatively estimate apoptosis in a population of these cells. Cytometry Part A 56A:89–95, 2003. © 2003 Wiley‐Liss, Inc.

Death receptor ligation triggers membrane scrambling between Golgi and mitochondria

“…19 To evaluate global aspects of endomembrane traffic, we exploited diverse fluorescent derivatives of Helix pomatia agglutinin (HPA), a lectin that recognizes membrane glycoproteins in the ER and proximal Golgi, 20 as well as at the surface of lymphoma cells. 21 The latter aspect reflects the traffic of glycoproteins that is modified during apoptosis, in part to stimulate binding of clearing cells. 22 Static HPA staining of internal membranes partially overlapped with ERGIC53-positive membranes, especially in the 'peri-Golgi' region, and likewise showed dispersal soon after , were acquired by using a state-of-the art Deltavision RT system coupled to an automated Olympus IX71 microscope with a  100 objective.…”

“…Mitochondria and other organelles were specifically stained either in solution (for intensified video microscopy (IVM) analysis 37 ) or after attachment to polylysine-coated coverslips 35 with Mitotrackers (MTR green or red) and fluorescent lectins. 21 After 10-60 min incubation, cells were fixed with 4% paraformaldehyde (w/v in PBS), permeabilized with Triton-X100, blocked, incubated with monoclonal antibodies for 1 h, washed again and then incubated for 45-60 min with anti-mouse secondary antibodies conjugated with Alexa Fluor 488 or Rhodamine X. See figure legends for details of fluorescence microscopy imaging.…”

“…Endocytosis was evaluated following the internalization of fluorescent HPA, which binds to surface glycoconjugates of Tlymphoma cells. 21 Measurements were carried out in a plate reader with appropriate filters (Fluoroskan Ascent, Thermo, Basingstoke, UK). Endocytosis was also measured following the fluorescence changes of FM1-43 35 in a plate reader, using 5 Â 10 5 cells/ml in a modified Ringer buffer (RB, containing 145 mM NaCl, 4.5 mM KCl, 2 mM MgCl 2 , 1 mM CaCl 2 , 5 mM K-HEPES, pH 7.4, and 10 mM glucose) and 2-4 mM FM1-43.…”

Sweet taste of cell death: role of carbohydrate recognition systems

“…In 2003, we [30] and another group of investigators [31] independently proposed a similar lectin-based approach for detecting apoptotic cells which demonstrated changes in GPs pattern of PM at apoptosis. For comparing an expression of PM glycoproteins in intact and apoptotic cells, we have used a big set of peroxidase-and fluorescent dye (FITC)-labeled lectins.…”

“…splenocytes, murine L1210 leucocytes inoculated to mouse) grown cells were studied (26,30). Changes in cell PM glycoepitops were detected with peroxidase-labeled specific lectins with subsequent cytochemical analysis and of FITC-labeled lectins followed by flow cytometry.…”