(all media and solutions used in cell culture are pre-warmed in a water bath before use!)
- Remove culture med
- Wash with pre-warmed DPBS (without Mg2+ and Ca2+)
- Add 2ml trypsin/T75 or 1ml trypsin/T25 flask and incubate until all cells are round (insufficient trypsinization will hinder downstream processes, and facilitates cell aggregation during implantation)
- Gently tap the side of the flask to dislodge remaining cells.
(if more than gentle agitation is required the trypsinization was incomplete and might hinder downstream processing, such as aggregation during injection)
- Add up to the original culture volume of complete medium.
- Pipette up/down thoroughly to shear cell clumps into single cell suspension. (do not generate foam during this process as foam is indicative of mechanical shearing of the cells)
- Transfer into a sterile 13ml tube and centrifuge for 4 minutes at 200 x g at room temperature.
- Aspirate supernatant and add 1 ml sterile PBS.
- Carefully resuspend the cells using a sterile 1000ul tip.
- Remove 10ul cell suspension for counting and transfer the remaining cell suspension to the centrifuge.
- Centrifuge for 4 minutes at 1200 x g at room temperature.
- Remove all PBS and leave to stand for 2 minutes to allow the remaining PBS clinging to the walls of the tube to settle, completely remove all PBS from the cell pellet.
- Dilute the cells to 250 cells /nl in 2% PVP40 as follows:
- Cell concentration (in 106)/(250 cells/nl) *1000
- Example : (2,5×106 cells/(250 cells /n)l *1000=10ul)
- Thoroughly resuspend the cells, while preventing the formation of air bubbles.