First question here so bear with me if something is unclear:
Would anyone have an idea as to wether it is possible to extract RBCs (from fish) that has
a) dried up in an EDTA vacutainer for several months (allthough kept refrigerated)
b) clotted/clumped up in the same sort of vacutainer but for approximately 10 weeks.
I've managed to break everything free by adding heparine, but there's still too much debris in the samples, both large pieces of what I assume is coagulating proteins.
I'll have to stain (the nuclei) with Propidium Iodide before sending them through the FCM, and that's where the debris becomes a real problem as it clogs the apparatus.
I'm guessing the best chance is to isolate the cells that may still be whole somehow? Problem is that there's so much "else" in the samples and I have no idea at which end to begin.
If the blood has clotted and dried up, heparin will have limited effect in dissociating the cells. Have you thought of digesting the fibrin? You may want to try urokinase or streptokinase. Good luck
Siva
Hi Erlend
In addition to Muttuswamy's suggestion you might consider gently centrifuging the cell suspension through a layer of high protein (e.g. bovine serum) or sucrose. Providing the intact RBCs are heavier than the debris, the RBCs will pass through the viscous solution faster than the debris. It will not give a perfect result but might help to clean up your sample.
You need to be mindful that active enzymes in your cell suspension may cleave RBC membrane molecules as well, so need to consider this for FCM if you are intending to look for specific RBC surface antigens.
Good luck, Rosemary
Thank you both so so much!
I will try out your suggestions, and hopefully my results will improve.
Oh: I'm not using the FCM to look for antigens, but to look at the nuclei size after staining it with Propidium Iodide. But as long as I can clean out the larger debris then the largest obstacle is gone at the very least.
Again, thank you :)
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