Hello,
I am optimizing a flow cytometry experiments for hACE2 using an RND systems polyclonal goat antibody (0.25ug/1Mcells same as spec sheet and already tried high and low concentrations, to no avail) and Alexa647 anti-goat secondary (1:400). I am testing A549 ACE2 overexpressing cell line, 293FT, 293FT ACE2 overexpressing stably transfected line. They are the same constructs and expression confirmed by Western. I am observing high if not the same signal in 293FT (negative control) vs transfected positive control. I noticed my IgG for all lines was high so I blocked with 2% human serum with or without 1% BSA and got the same peaks at 10^4. I am now following a brief protocol below:
Collect cells, PBS wash, block with 1% BSA 30min at RT. Spin wash 1x. Primary 1hr 4C in 1%BSA. 2x wash. Secondary ab 30min RT. 2x wash and fix in 2% PFA for 10min then 2x wash and store 4C then run next day.
Maybe reduce primary incubation time? Any help would be very much appreciated!
Thanks,
Dale
Hello Dale Allen ,
Has your polyclonal Ab been tested before in Flow Cytometry (published article or tested by manufacturer). You might need to get in contact with RnD Systems to verify that. I faced recently something similar to that, where the ventor contacted me to let me know that the batch I got was not recommended for Flow Cytometry, as there was a lot of non-specific binding. The specificity though was still in place when tested in WB.
Another thing I noticed is that you're using a secondary antibody from different species than the serum used for blocking. I would also try to block with goat serum (alone or in combination with the species of primary Ab). In addition, due to the subsequent staining with another Ab, I would have tried to use it alone, if you haven't do it so far, to evaluate any background noise or non-specific binding.
I guess you have already checked the PMT voltage in the APC channel (preferably with cells, not just beads). Sometimes we might overdo and mistakenly set it high, thus the negative population shifts (in case your antibody is not functional at all in FACS that might be also the case).
Good luck!
Best
Filip
I reran this with only 30min RT primary incubation and the background was decreased. Hope this helps anyone. Yes the antibody was tested published and validated for Flow. Thanks for your response Filippos!
because HEK293 cells were derived from human embryonic kidney cells, they might have background constitutive expression of hACE2. This might explain high background in some readouts.
The following method might allow you to quantitate ACE activity for the over-expressing cell line as well as the parental one. Use inhibitors to see how much of it could be inhibited in the parental cell line. https://pubmed.ncbi.nlm.nih.gov/28116710/
Additionally, there might be several options out there on how to use pharmacological agents linked to GFP or to fluorochromes that can be used in imaging or flow cytometry. Article Fluorescence imaging of drug target proteins using chemical probes
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