1) Researchers developed a cell-free system to induce apoptosis in isolated chicken liver cell nuclei similar to how it occurs in intact cells. The nuclei underwent morphological changes characteristic of apoptosis like chromatin condensation and DNA fragmentation within 60 minutes. 2) Electron microscopy was used to observe the ultrastructural changes during apoptosis and necrosis in different cell types, like nuclear fragmentation and chromatin condensation during apoptosis. 3) A study compared analyzing cell death using annexin V/propidium iodide staining flow cytometry to holographic microscopy coupled with fluorescence detection, finding the latter better identified oncosis due to ability to observe morphological changes.

1. 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.
Both processes are inhibited by Zn++ in intact
cells
Chormatin highly condensed spherical domain
Nuclear DNA cleaved in Nucleosomal
ladder
Excrude throgh Nuclear envelope forming
apoptotic bodies structural changes occur
in 60 min
Prepared from Mitotic Chicken Hepatoma cells following a sequential S/M phase
synchronization
Nuclei Added to these extract

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

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)

8. 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.

10. Red arrow—nuclear
fragmentation
Light blue arrow—chromatin
condensation

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

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.