Brief description of cell cytotoxicity assays. I have uploaded a separate ppt on cell viability assays. Prepared in July, 2015

1. Ashikh Seethy
Junior Resident
Dept of Biochemistry
Maulana Azad Medical College
New Delhi
Cell Cytotoxicity Assays

2. Overview
 Cell Death
 Apoptosis
 Assays for Apoptosis
 Other cytotoxicity Assays

3. Cell Death
 No of cells in human body?
 37 trillion human cells
 Cells death Vital for homeostasis
 Diseases
 Cell Death:
 Type I: Apoptosis
 Type II: Autophagy
 Type III: Necrosis

5. Apoptosis

6. 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

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

9. Mechanism of Apoptosis

12. Assays for Apoptosis:

13. Synthetic Substrates Assays

14. 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

15. Membrane Alterations:
 Phosphatidyl Serine
 Located in inner leaflet of plasma membrane
 Flipped to outer leaflet by Caspases
 Signal for Phagocytosis
 Binds with Annexin V
 Annexin V forms trimers on binding with Phopshatidyl Serine

16. Annexin V

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

19. 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.

20. TUNEL (terminal dUTP nick end-
labeling)
 Terminal
deoxynucleotide
Transferase

21. 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

22. Other Cytotoxicity
Assays

23. 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.

24. MTT Assay for Cytotoxicity:

25. MTT Assay for Cytotoxicity:
Seed the wells with the no. of cells corresponding to OD of 0.8 to 1.2
Incubate so as to enable cells to adhere and grow
Add the drug in serial dilutions to the wells
One set of wells act as control
Incubate for varying periods of time
Perform MTT Assay
Calculate the ODs and their average
• Cell Death (%) = 100 - [ODTx100/ ODC]
• From this IC50 can be calculated

27. Thank You