Preparing cell suspensions for flow cytometry

Reference no: EM133135659

BC3102 Molecular Basis of Disease - James Cook University

Practical: Preparing cell suspensions for Flow Cytometry

Learning objective 1: Demonstrate accurate and precise pipetting skills which will be needed to complete the practical exercise; Prepare a splenic single cell suspension

Learning objective 2: Accurately determine cell number and viability using different cell counting methods

Learning objective 3: Understand the principles of Flow cytometry and identify on the basis of antibody staining, the proportions and numbers of cells within the major subsets of splenocytes.

You will interpret the data produced by the flow cytometer from analysis of a sample stained with fluoresceinated monocloncal antibodies.

Question 1. What is the importance of the MACS buffer?

Question 2. Why do you want to ensure that no ice gets into the petri dish when making the cell suspension?

Question 3. What is the purpose of the RBC lysing buffer?

Question 4. What was the cell concentration of your cell suspension from the Coulter counter (show your working)?

Question 5. What was the total leukocyte numbers in the spleen (show your working)?

Question 6. What was the volume required to pipette 1x106 cells of your cell suspension (show your working)?

Question 7. What proportion of cells were viable?

Question 8. What was the cell concentration of your cell suspension from the Countess cell counter (attach your printout)?

Question 9. What was the total leukocyte numbers in the spleen (show your working)?

Question 10. What was the volume required to pipette 1x106 cells of your cell suspension (show your working)?

Question 11. What proportion of cells were viable?

Question 12. What was the cell concentration of your cell suspension using the Haemocytometer (show your working)?

Question 13. What was the total leukocyte numbers in the spleen (show your working)?

Question 14. What was the volume required to pipette 1x106 cells of your cell suspension (show your working)?

Question 15. What is the purpose of cell counting?

Question 16. Why is cell counting important?

Question 17. What is the principle of a cell counter?

Question 18. What is the principle of automated impedance cell counters?

Question 19. What is the greatest limitation of automated cell counters?

Question 20. What is manual counting?

Question 21. Which method of cell counting is more accurate?

Question 22. What is the FSC-H vs FSC-A plot used for (see the results data)? Explain what this plot does

Question 23. What is the goal of a titration? -particularly with respect to the positive and negative populations?

Question 24. Which Ab titration provided best staining of CD4 T cells?

Question 25. Which Ab titration provided the best staining of CD8 T cells?

Question 26. What proportion of cells were CD4 T cells?

Question 27. What proportion of cells were CD8 T cells?

Question 28. How can you use a plot of side scatter (SSC-H) versus forward scatter (FSC-H) to identify blood cell populations? Use your knowledge on how cell complexity affects cell distribution on the plot to answer this question.

Attachment:- Flow Cytometry.rar

Reference no: EM133135659

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Reviews

len3135659

4/29/2022 4:42:59 AM

hi, I need you help for this assignment. The dilution factor for coulter counter and contess is 1/500. The dilution factor for countless is 1/200. you need answers to all the questions. The formula to find cell concentration and to find total no. of leukocytes are given in the practical FILE for question 22-28 ia attached it consist flow cytomter charts. The haemocytomreter cell count is 109. and dilution factor 1/200. Add this image where printout is asked. If you need any help please let me know.

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