FACS & Cell Sorting

1. FACS & Cell Sorting Cell Isolation 1- FACS (fluorescence-activated cell sorter) 2. By density gradient: Ficoll-Hypaque, Percolletc 3- By antibody-based methods other than magnetic beads & FAC 4- Bymagnetic beads

3. NECROSIS C B A APOPTOSIS

4. Thio proteases

5. Detection of Apoptosis by Flow Cytometry • Early stage Annexin V/7-AAD(PI) • Mid stage TUNEL assay • Late stage < Go/G1 DNA content

6. B C A Apoptosis D F E

9. Annexin V The translocation of PS precedes other apoptotic processes such as loss of plasma membrane integrity, DNA fragmentation, and chromatin condensation. As such, Annexin V can be conjugated to biotin or to a fluorochrome such as FITC, PE, APC, Cy5, or Cy5.5, and used for the easy, flow cytometric identification of cells in the early stages of apoptosis.

10. Annexin V Because PS translocation also occurs during necrosis, Annexin V is not an absolute marker of apoptosis. Therefore, it is often used in conjunction with vital dyes such as 7-amino-actinomysin (7-AAD) or propidium iodide (PI), which bind to nucleic acids, but can only penetrate the plasma membrane when membrane integrity is breached, as occurs in the later stages of apoptosis or in necrosis.

11. Annexin V No Apoptosis = Cell ViabilityCells that are negative for both Annexin V and the vital dye have no indications of apoptosis: PS translocation has not occurred and the plasma membrane is still intact.Early ApoptosisCells that are Annexin V-positive and vital dye-negative, however, are in early apoptosis as PS translocation has occurred, yet the plasma membrane is still intact.

12. Annexin V Late Apoptosis or Cell DeathCells that are positive for both Annexin V and the vital dye are either in the late stages of apoptosis or are already dead, as PS translocation has occurred and the loss of plasma membrane integrity is observed.When measured over time, Annexin V and a vital dye can be used to monitor the progression of apoptosis: from cell viability, to early-stage apoptosis, and finally to late-stage apoptosis and cell death.

13. 4.72 56.69 93.82 41.84 1.46 2.37 61.22 12.67 37.02 85.25 2.08 1.76

16. Group of cysteinyl-aspartic acid proteases is called caspases. Caspases have been divided into three groups based on the four amino acids amino-terminal to their cleavage site Asp-Glu-Val-Asp DEVD

17. to 7-amino-4-trifluoromethyl coumarin

18. offers an anti-PARP-FITC conjugated Cleavage Site-Specific Antibody (CSSA) that can detect apoptotic cells by flow cytometry. Dr Atef Masad

19. Exogenous Terminal deoxynucleotidyl transferase (TdT )is used to catalyze a template-independent addition of bromodeoxyuridine triphosphates (Br-dUTP) to the free 3’-hydroxyl ends of double or single stranded DNA fragments. This can be identified by FITC conjugated anti-Bromodeoxyuridine (BrDU) antibodies Then tthese antibodies can be analyzed using a flow cytometer or a fluorescence microscope. Dr Atef Masad

20. Dr Atef Masad

21. Dr Atef Masad

22. TUNEL staining relies on the ability of the enzyme terminal deoxynucleotidyl transferase to incorporate labeled dUTP into free 3'-hydroxyl termini generated by the fragmentation of genomic DNA into low molecular weight double-stranded DNA and high molecular weight single stranded DNA Dr Atef Masad

23. TUNEL staining may also be used to detect DNA damage associated with non-apoptotic events such as necrotic cell death induced by exposure to toxic compounds and other insults , and TUNEL staining has also been reported to stain cells undergoing active DNA repair Dr Atef Masad

24. Dr Atef Masad