I was wondering if I would be able to use BV2 cell line to set up the compensation controls for apoptosis assay on flow cytometer. I am staining the cells with annexin and PI.
I would like to measure apoptosis in BM murine neutrophils, but I would like to reduce the number of animals and resources to get the neutrophils population while I'm still working out the protocol issues for the assay
i need to figure out the positive and negative controls. How they read on the flow, and make sure these controls would work as the compensation controls. I was wondering what are the down side of using a cell line like BV2 cells or Raw cells in doing so
also, if I am successful to generate these controls from the cell lines, could I use them as the compensation controls if my actual experiment will be on neutrophils?
Thank you
In my experience, compensation and voltage settings for different dyes are dependent on the cell type you are analyzing on flow cytometer. Thus, I would recommend using neutrophils rather than BV2 cell line for compensation and fluorescence minus one (FMO) controls in the real experiment. However, to reduce amount of animals while working on the protocol, you can use a cell line to set up the parameters and check the whole procedure. Once you have the protocol running, I would do initial experiment with neutrophils with all compensation and FMO controls to optimize it for neutrophils. Depending on the instrument (how good it is maintained) and if you do not change parameters of your staining procedure and staining panel, the instrument set-up should be rather stable and you will only need to periodically check it. I hope this helps.
In my experience, compensation and voltage settings for different dyes are dependent on the cell type you are analyzing on flow cytometer. Thus, I would recommend using neutrophils rather than BV2 cell line for compensation and fluorescence minus one (FMO) controls in the real experiment. However, to reduce amount of animals while working on the protocol, you can use a cell line to set up the parameters and check the whole procedure. Once you have the protocol running, I would do initial experiment with neutrophils with all compensation and FMO controls to optimize it for neutrophils. Depending on the instrument (how good it is maintained) and if you do not change parameters of your staining procedure and staining panel, the instrument set-up should be rather stable and you will only need to periodically check it. I hope this helps.
I agree with Berislav.
You can use a cell line to set up the voltages for detection of PI and AV. If you are only doing apoptosis assay without measuring additional markers, you can avoid compensation by choosing AV-APC/FITC/PE and replace PI with DAPI. FMO is required if the neg and pos populations are not distinct. An unstained control prepared from a healthy control mouse or cell line should be sufficient. Positive control is damaged cells. You can prepare this by adding few drops 70% ethanol into the cells>spin>ressuspend.
You will need to adjust the FSC-A and SSC-A for neutrophils because these parameters vary depending on the cell population you measure. But the voltages should remain the same for PI and AV as long as you use the same dilution you optimized with the cell line.
(1) According to the compensation principle and my personal experience, It is totally ok to use different cells or beads to control compensation if you can play both control and target cells in the same parameters settings.
(2) The ideal controls are those mixture of clearly resolved negative and positive two populations.
(3) There are three ways to do compensation: A. on-machine manual change voltage; B get the compensation matrix by acquisition software; C compensate by analysis software, such as Flowjo.
Berislav,
Thank you for your explanation.
thats exactly what i needed!!
Kai Ling,
I will be using the annexin V/ Pi apoptosis kit. we already purchased it and I have done some experiments with it.
you are right, you do need the light scatter parameters adjusted for your cells but the voltages for the fluorescent channels can stay the same.
thank you for your suggestion for the positive control
It sounds easy to use. it would be helpful.
Shiqiu,
Thank you for your suggestion,
i do the compensation on Flowjo
I am going to try to use neutrophils for compenation, but its good to know i can use the cells and the beads
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