I'm wondering what kind of integrity the cells have afterwards. I'm using cell tak to adhere bovine lymphocytes and then interrogating their OCR and ECAR with the seahorse instrument.
Thanks for you input, but I'm actually looking for the reverse. I've been using CellTak to adhere suspension cells and I would like to disengage them from the plate and use them for further analysis such as flow or microscopy. I was wondering if Cell Tak alters the cells anyway or if there is a viability issue afterwards.
Hey Jordan,
Have you had any luck with this? I use cell tak for my cells and I tried to perform a BCA on my cells the other day using my normal method of pipetting up and down a few times then transferring to a epee to incubate. But... The end result was no difference in protein content compared to the blank well. And I seeded 1 million cells per well! So obviously none of the cells disengaged with the pipetting. So obviously it is pretty strong.
I was going to try next incubating in the plate with the buffer to see if it would just burst the cells open while they were still attached to the plate.
I also read something about trypsin being used to remove the cells but I can't remember where I read that now. Sorry! But that also might shear off any cell surface antigens you are staining during FACS. But something to try...
I have also contacted CLS to see if they can suggest something to detach the cells once I have finished with my assay.
Let me know how you go!
Hi Helen,
I used trypsin to remove my cells (bovine non adherent lymphocytes from PBMCs) and they came off great with ~99% viability. But, with it being trypsin I don't know the extent of surface marker damage. Right now, I am using these cells to extract DNA for analysis, but plan to recover cells for flow at some point. Still looking for a "gentler" method of recovering cells and will keep you posted.
Jordan
Hi,
there is this review about several aspects of SeaHorse, at the end they discuss of hearvesting cells after SH assay (for normalization issue). They used trypsin as well, but only for cell count (and flow but without IF I suppose).
Ben
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Thank you! In order to save face with surface markers I have started using Hank's Buffer Saline Solution with 0.5% EDTA and incubating for 15-20min at 37C and then pipetting to get my cells. It seems to work relatively well. There still seems to be some loss of cell number, but it is a decent option.
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