I'm developing an adhesion assay with CFSE-labeled polyclonal T-cells. Basically, I allow the cells to adhere to a poly-l-lysine coated (0.01%) well in a 24 well plate, and after gentle washing, I read the fluorescence at an excitation of 494 and an emission of 517 nm.
Incidentally, the plate reader reports there being no signal, despite the fact that I can see cells when I look at the wells under the microscope. Should I increase the cell density? I am seeding 500,000 cells per well at this point. Or, is there something else that I am missing?
1. Did you QC your CFSE staining? I stained with CFSE and fluorescence was very strong.
2. Are you using compatible plates? For fluorescence assay you should use black plates or the signal could be influenced from adjacent wells. Using transparent plates could give rise to high background readings caused by reflection.
3. Are you using your flourometer properly? Optimized z position etc.
Hi Ehud!
Thanks so much for your prompt response! :)
1) I have totally QC'd my CFSE. From a microscopy standpoint, the cells do show strong fluorescence.
2) I am using transparent plates, but I always make sure to cover the bottom with foil, while skipping rows between samples to prevent bleed-over. I don't think that this is the issue since even my PBS blank gives a reading of zero.
3) I am not sure about the z-position et al. I am, essentially, using the BioTek Synergy HT plate reader whose fluorescence settings go as far as selecting your emission/excitation.
Once again, thanks so much!
Best,
A.
Hi Akshat,
4. Have you tested cells fluorescence before adhering? Soon after dispensing into the wells. This should also be a good positive control.
5. Are you sure the cells you observe are viable? If they are dying or disrupted as result of poly lysine conjugation their content could be spilled and diluted.
Regards.
E.
Ehud,
4) That is an excellent point! I should definitely check that out.
5) The viability question is also something that I have indirectly addressed wherein I looked seeded the cells without poly-l-lysine in parallel with a coated plate, and got the same zero fluorescence results.
Thanks!
Best,
A.
Hi Ehud!
So, I did the assay today in non-adhesion conditions, and actually got some pretty good/expected signals from the CFSE labeled cells. I am now beginning to wonder if perhaps the poly-l-lysine coating might be the problem, or perhaps, fixing the cells with glutaraldehyde after washing might be the culprit?
What do you think? I'd be very grateful for your input!
Best,
A.
Hi Akshat,
I think Glutaraldehyde is a very reactive molecule, I know it is used in protein conjugations (via amine residues) I would put my money on it.
If I remember correctly you could fixate your cells with formaldehyde as well, it might do the difference.
I don’t know about polylysine reactivity.
Should be easy enough to test, check for fluorescence after every major step you do, And you would find your culprit. (Hope your fluorometer is not too far).
I am not sure what you are trying to achieve with plate bound CFSE stained T cells.
Be glad to help further.
Regards,
Ehud
Hey, Ehud!
Thanks so much! I'll definitely do this, and see what I find.
Well, what I am doing right now is trying to optimise an adhesion assay, that I will eventually use to ask questions about T-cell and NKT cell functional responses to different concentrations of integrins/ the presence and/or absence of different ECM components. But, I need to get past the initial hurdles of getting the assay to work first! :)
Once, I have the fluorescence issue sorted, I'll start looking at means to bind ICAM-1-Fc to the plates.
You've been such an excellent resource, and for that, I am most grateful!
Thanks a lot, sir!
Best,
A.
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