Cells undergoing morphological changes during apoptosis

Cell culture- Chicken DU249 Hepatoma cell grown in RPMI 1640 plus 10% FBS in Monolayer or suspension culture preparation of S/M Extract.
Microscopy- Fluorescence microscopy was performed using an Olympus Vanox microscope equipped with a filter set from chromo technology corporation.
Images were acquired using a DAGE SIT camera controlled by Hyperscope image analysis program.
Staining Technique - MPM-2STAINING
Murine thymocyte treated with 0.1uM dexamethasone were attached to Adhesio-Slides . SN12C cells were grown on coverslip.
Cells were fixed 4% formaldehyde processed for indrec immunofluoresence using MPM-2 antibody diluted 1:200 and biotinylated horse anti-mouse secondary antibody with streptavidin-texas red.

1. Cells Undergoing Morphological changes during apoptosis By Vivek Sharma AND NAVEEN SINGH Roll No.20ppc001

2. Yuri A Lazebnik et al developed a cell free system induce morphological transformation characteristics of Apoptosis in isolated nuclei.

3. Cell culture- Chicken DU249 Hepatoma cell grown in RPMI 1640 plus 10% FBS in Monolayer or suspension culture preparation of S/M Extract. Microscopy - Fluorescence microscopy was performed using an Olympus Vanox microscope equipped with a filter set from chromo technology corporation. Images were acquired using a DAGE SIT camera controlled by Hyperscope image analysis program. Staining Technique - MPM-2STAINING Murine thymocyte treated with 0.1uM dexamethasone were attached to Adhesio -Slides . SN12C cells were grown on coverslip . Cells were fixed 4% formaldehyde processed for indrec immunofluoresence using MPM-2 antibody diluted 1:200 and biotinylated horse anti-mouse secondary antibody with streptavidin-texas red.

4. Similar morphology of nuclei from human GM3798 lymphoblastoid cells undergoing apoptosis in vitro and in vivo. (A) Apoptosis in living GM3798 cells was induced by addition of 0.1 um dexamethasone to growing cultures. This cell entered apoptosis 6 d after addition of the drug. (B) Nuclei isolated from GM3798 cells were placed in S/M extract and incubated for 1 h at 37°C before fixation for EM.

5. The nuclear lamina is dissembled in cells undergoing apoptosis in culture. (A'-A ~) Chicken hepatoma DU249 cells, which occasionally undergo spontaneous apoptosis in culture. (B'-W) Thymocytes from C57B1/6 mice following exposure to 1/~M dexamethasone in culture. (C'-C') A portion of these cells that were cultured in the absence of dexamethasone . (D-D') Human SN12C renal carcinoma cells undergoing apoptosis in culture in response to the sequential exposure to camptothecin and tumor necrosis factor. (A-D) phase contrast; (A'-D') DAPI staining of the DNA; (A~-D ~) visualization of lamin A using an affinity-purified antibody directed against a peptide determinant. The arrowheads in the latter panels indicate cells undergoing morphological apoptosis

6. Multimodal Holographic Microscopy: Distinction between Apoptosis and Oncosis Given by Jan Balvan et al.

7. Identification of specific cell death is of grat value for many scientist..predominant types of cell death can be detected by flow cytometry (FCM). the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis and the definition of the oncosis is important because of its potential reversibility. FCM analysis of cell death using annexin V/ propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - “Multimodal holographic microscopy (MHM) Jan Balvan et al.

8. Holographic microscope The light is divided into two separate optical paths—object arm and interferometer reference arm. Both arms consist of condenser (C), objective (O) and tube lens (TL). In the reference arm, a diffraction grating (DG) is placed. The object beam and the reference beam recombine in the output plane and create interference fringes pattern, which is captured by the camera (D). S—source; CL—collector lens; BS—beam splitter; M—mirror; C—condenser; O-objective; TL—tube lens; DG—diffraction grating; OL— output lens; D—detector.

9. Halographic microscopy images MHM 20x

10. TEM IMAGES A(2800x) and D(5600x) Red arrow—nuclear fragmentation Light blue arrow—chromatin condensation

11. Analysis of Cell Death by Electron Microscopy S. Burattini and E. Falcieri

12. Cell death is said to occur mostly by two alternative, opposite modes, Which involve a highly genetically regulated and elaborate network of biochemical events and cascades , and necrosis, consider a passive cell death without underlying regulatory mechanism. They describe different morphological changes of cells undergoing apoptotic and necrotic cell death. TEM allow detailed sudies of ultrastructural changes,with in cell such as nuclear alteration. The cell surface changes including membrane blebbing and loss of features , such as microvilli,can be assessed by SEM Study by S.Burattini

13. SEM of fl oating ( a , b ) and adherent ( c , d ) cultured cells. CD34 stem cells in apoptosis ( a ): several blebs are visible. K562 erythroleukemia human cells incubated with natural killer cells: the appearance of membrane discontinuities is shown. UVB-treated C2C12 myoblasts ( c ) and myotubes ( d ): a diffuse blebbing characterizes apoptotic death. ( a ) Bar = 1 μ m. ( b , c ) Bars = 0.2 μ m. ( d ) Bar = 10 μ m

14. Lazebnik , Yuri & Cole, Susan & Cooke, C & Nelson, W & Earnshaw , William. (1993). Nuclear events of apoptosis in vitro in cell-free mitotic extracts: A model system for analysis of the active phase of apoptosis. The Journal of cell biology. 123. 7-22. Burattini , S. and Falcieri , E., 2013. Analysis of cell death by electron microscopy. In  Necrosis  (pp. 77-89). Humana Press, Totowa, NJ. Balvan , J., Krizova , A., Gumulec , J., Raudenska , M., Sladek , Z., Sedlackova , M., Babula , P., Sztalmachova , M., Kizek , R., Chmelik , R. and Masarik , M., 2015.Multimodal holographic microscopy: distinction between apoptosis and oncosis .  PloS one ,  10 (3), p.e0121674. REFRENCES