Cell cytotoxicity assays

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

4. Apoptosis

5. 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

7. Mechanism of Apoptosis

8. Assays for Apoptosis:

9. Synthetic Substrates Assays

10. 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

11. 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

12. Annexin V

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

14. 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.

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

16. 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

17. Other Cytotoxicity Assays

18. 51 Cr Release Assay: Target cells are first incubated in a solution of sodium 51 chromate, 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 51 Cr into the supernatant of the mixed cell culture Quantified by comparing the 51 Cr release from the test cells with that from detergent treated and control cells.

19. MTT Assay for Cytotoxicity:

20. 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 - [OD T x100/ OD C ] From this IC 50 can be calculated

21. Thank You