My cells are forming a rare clump during harvesting. I harvest at 80 to 90% confluence using 1X trypsin (0.5 g porcine trypsin and 0.2 g EDTA, 4Na per liter of Hanks' Balanced Salt Solution with phenol red). After approximately 5 to 6 minutes, I check my cells, and they are all round and unattached to the plastic. I inactivate the trypsin with medium containing 5% FBS and then centrifuge at 1000 rpm for 8 minutes. Here is where I encounter the problem: the cells form a transparent, non-pelleting clump. This structure remains in the upper part of the supernatant and is extremely viscous, sticky and impossible to break with 1ml pipete. I do find a smaller white pellet in the precipitate, but most of my cells are trapped in the upper clump, and I am losing time and process efficiency. There should be 100 million cells in this suspension, but I'm barely finding 30 - 50 million.
I've worked with these cells and protocols before, and I've never encountered this problem. I don't know if I am not flushing enough or manipulating incorrectly during inactivation. A colleague used the same steps, and the harvest was successful.
Thanks in advance.
It is likely that you have a clump of chromatin/DNA from lysed cells. If you add some DNAase to the dispersal medium this will be minimize. Also be sure to use Calcium/magnesium free Hanks' . A 10 minute rinse in CMF Hanks' at the beginning will also help. Also avoid bubbling, frothing when pipetting. The surface tension of the bubbles lyses cells.
https://www.stemcell.com/products/dnase-i.html#:~:text=Deoxyribonuclease%20I%20(DNase%20I)%20is,a%20result%20of%20cell%20damage.
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011904_Dnase_I_RNasefree_UG.pdf
https://medicine.uiowa.edu/flowcytometry/disaggregation-clumped-cells
- Badges
- Science topic