FACS Flow cytometry| Apoptosis & Necrosis| CSIR NET SET Questions
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The document discusses the use of flow cytometry and Annexin V-FITC to identify apoptotic cells by detecting phosphatidylserine (PS) translocation during apoptosis. It highlights the role of propidium iodide (PI) in distinguishing necrotic and late apoptotic cells, and poses questions regarding cell populations in specific quadrants of a FACS analysis plot. Additionally, it mentions a researcher’s confusion in labeling samples during an experiment and asks about ligand binding affinity of various receptors.
FACS Flow cytometry| Apoptosis & Necrosis| CSIR NET SET Questions
- 2. CSIR NET DEC 2012 2 During apoptosis, phosphatidylserine(PS) usually present in the inner leaflet of the plasma membrane flips to the outer membrane. Annexin V is a protein that finds PS, using this as a tool, we identify the apoptotic cells from necrotic and normal cell populations by FACS using FITC- tagged Annexin V. Propidium iodide (PI) is used to stain the nucleus which generally identifies necrotic and late apoptotic cells. In which area of the plot you should get early apoptotic cells by FACS analysis
- 3. 3 PHOSPHATIDYL SERINE (PS) PS is negatively charged Annexin V Ca2+ phospholipid binding protein Annexin V conjugated with FITC Can’t distinguish between apoptosis and necrosis!
- 4. ➔ Cationic dye ➔ Not taken up by live cells ➔ Apparent only in the late phases of apoptosis 4
- 5. FACS RESULTS 5 PI Annexin V-FITC A P A P A P A P Live cells Early apoptosis Late apoptosis Necrosis
- 6. CSIR NET DEC 2012 6 During apoptosis, phosphatidylserine(PS) usually present in the inner leaflet of the plasma membrane flips to the outer membrane. Annexin V is a protein that finds PS, using this as a tool, we identify the apoptotic cells from necrotic and normal cell populations by FACS using FITC- tagged Annexin V. Propidium iodide (PI) is used to stain the nucleus which generally identifies necrotic and late apoptotic cells. In which area of the plot you should get early apoptotic cells by FACS analysis A. Quadrant I B. Quadrant II C. Quadrant III D. Quadrant IV
- 7. CSIR NET DEC 2014 7 A researcher was repeating a FACS experiment but somehow got confused with the labeling of the tubes. There are four tubes, on control, C(with no fluorescent label), one standard 1, S1(with FITC label), one standard 2, S2(with PE label) and the last one test, T(which should be FITC positive). Given below is the result of the FACS experiment.
- 8. 8 Surface Antigen Anti body Dye A Anti-A Fluorescein GREEN B Anti-B Rhodamine RED DEC 2019
- 9. 9 Which receptor has the highest ligand binding affinity? a. A b. B c. C d. D