Flow Cytometry Setup Help!

[bme3257]

Hey Everyone!

I have no experience with flow cytometry and I have been presented with this experiment that I have to set up- can anyone please help me out with this?

Design a flow cytometry experiment to characterize Human Lymphocytes [both major and minor subsets]



A. Using the available pool of CD markers, choose the combinations which you think are the best (justify) and design two 5-8 color panels to characterize/estimate the following: [each panel can be dedicated towards characterizing one of the following]



· General lymphocyte subsets (T [helper and cytotoxic T cells], B and NK Cells)

· T cells minor subsets (activated/naive/memory T cells)



B. Also list all the control tubes (negative/compensation controls) you will prep for this experiment.

C. From the panels you have designed, list what combinations of CD markers you will use to identify each major and minor cell subsets. Describe a gaiting strategy for the above panels to gate the population of interest and estimate the percentage of each subset present.



Available configuration of the instrument is: (4-Laser, 14 Parameter, including FSC and SSC)

Optical filters to detect the following fluorophores:



1. Blue laser (488nm)

SSC

FITC(505LP; 525/50, rb532)

PerCP Cy5.5 (690LP; 695/40)



2. Red laser (647nm)

APC (670/30)

Alexa 700 (685LP; 730/45)

APC CY7 (740LP; 780/60)



3. Yellow-Green Laser (561nm)

PE (582/15)

PE TxRd (595LP; 610/20)

PE Cy5 (635LP; 670/30)

PE Cy5.5 (690LP; 695/40)

PE CY 7 (750LP; 780/60)



4. Violet Laser (405nm)

Pacific Blue/Horizon V450 (450/50)

AmCyan/Horizon V500 (525/50)





You can use a combination of any of the above colors to design you assay. Remember to choose them in such a way that they have minimum spectral overlap


I appreciate your help!