I am trying to create a workflow for flow cytometry experiment sample prep. I intend to use LIVE/DEAD™ Fixable Aqua Stain and an antibody cocktail for extracellular antigens.
I intend to use the viability dye first. The protocol I found does not seem to mind the protein content in buffers, as the cell washing is performed with PBS (5-10% fetal cell serum(I imagine they meant "calf")), and the cells are suspended in the same buffer before viability staining. I read that the presence of proteins in the buffers might contribute to a higher background, is that correct? How should I adjust the solution (for washing and resuspension) if that is correct?
Additionally, I intend to perform antibody titration for the antibodies I'm using. There are a few questions here too. We have no prior experience with these antibodies in the lab. Questions:
1. Is it important to do titration for every antibody in the cocktail?
2. Do I keep the same general sample prep with the antibody cocktail, only swapping the antibody cocktail with a single antibody that's in different concentrations? Do I also apply the viability dye as well?
3. The antibody cocktail I am to use includes dyes such as SB and BV etc, which require a special staining buffer or blocking buffer. Do I need these special staining buffers or blocking buffers for titration as well? Is it necessary since titration typically only uses one dye at a time (together with viability dye?)?
I know the information above might not be detailed enough but could you share your personal experience related to these scenarios? The protocol that I base my protocol on comes from this page
- https://www.abcam.com/protocols/flow-cytometry-for-intracellular-and-extracellular-targets
Thanks a lot for your input!
Hello,
You can do your viability staining step as the first step of your FC staining, at the same time as your CD16/CD32 blocking step. You don't have to use fetal calf serum or BSA at this point.
For your other questions:
1. Titration is an important step if your plan to use your antibody panel often. Usually, the concentration proposed by the supplier is in the correct range, but the cell you used and the supplier's are for sure different, hence, antibody concentrations might be in need of adjustment.
2.You can prepare a master mix of antibody and prepare a serial dilution of this master mix. You don't have to prepare different dilutions of 1 antibody while the rest are the same, and repeat this step for each antibody. Titration you antibodies with your complete panel, this way you can take in account the signal leak for other fluophores. Titration of the viability dye is less important in my opinion. Supplier's proposed concentration for the viability dye should be enough.
3. Do your titration in the buffers you plan to use in your real experiment
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