Preparing cell suspensions for Flow Cytometry - Demonstrate

Assignment Detail:- 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

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