This document discusses various assays for detecting and measuring apoptosis (programmed cell death). It describes assays such as fluorescently labeled annexin V to detect phosphatidyl serine exposure on the outer membrane of apoptotic cells; caspase activation assays using antibodies against caspase substrates; assays measuring mitochondrial changes like cytochrome c release; TUNEL assay to label DNA fragmentation; and DNA fragmentation assays like detecting hypodiploid nuclei by flow cytometry. These assays allow researchers to study and quantify apoptosis in cells.

1. Assays of cell death

4. Apoptosis
 Induced by a tightly regulated
suicide program
 Cells destined to die activate
enzymes Degrade nuclear DNA
and nuclear and cytoplasmic
proteins
 Plasma membrane of the dying
cell is intact
 No inflammation

6. Role of Apoptosis
 Physiological
 Embryogenesis
 Hormone dependent tissues
 Proliferating cells
 Immune system
 Pathological
 DNA damage
 Misfolded proteins
 Infections

8. Assays for Apoptosis:

9. Fluorescently labelled annexin V measures
phosphotidyl serine in the outer lipid envelope of
apoptotic cells
 In cells undergoing apoptosis (programmed cell death), but not other modes of
cell death, the membrane phospholipid phosphatidyl serine flips from the interior
to the exterior side of the plasma membrane phospholipid bilayer.
 Annexin V is a protein that binds to phosphatidyl serine in a calcium dependent
manner.
 Fluorescently labeled Annexin V can therefore be used to tag apoptotic cells for
detection using flow cytometry or a fluorescent plate reader.

10. Annexin V

11. Caspase Activation:
 Detection of cleavage of known caspase
substrates
 Antibodies against PARP-1 [Anti-Poly (ADP-Ribose)
Polymerase]
 Antibodies that solely detect the caspase-
cleaved form, but not the native form
 Cleaved cytokeratin-15

13. Mitochondrial Changes
Cytochrome c assay:
 Western Blot after cell fractionation
 Immunofluorescence
 GFP-tagged cytochrome after selective
permeabilization of the plasma membrane by
digitonin  Extramitochondrial Cyt. C is removed
from the cell

14. 51Cr Release Assay:
 Target cells are first incubated in a
solution of sodium51chromate, which is taken up into the cells
 Excess chromium is washed out of the cell suspension
 The radioactively labelled targets are mixed with the killer cell
population at defined effector to target cell ratios
 Death of the target cells is indicated by the release of 51Cr into
the supernatant of the mixed cell culture
 Quantified by comparing the 51Cr release from the test cells with
that from detergent treated and control cells.

15. TUNEL (terminal dUTP nick end-labelling
 Terminal
deoxynucleotide
Transferase

17. DNA Fragmentation:
 Caspase Activated DNase (CAD)
 Creates fragments of 180 bp and its
multiples

18. DNA Fragmentation:
 Detection of hypodiploid nuclei
 Performed in a flow cytometer using DNA-binding
fluorochromes such as propidium iodide
 Cytoplasmic nucleosomes
 ELISA techniques using a combination of anti-
histone and anti- DNA antibodies.